A Dissertation entitled
Marinobufagenin Induced Uremic Cardiomyopathy: The Role of Passive Immunization, Rapamycin, and CD40 Signaling in The Generation of Renal Fibrosis
By
Steven T. Haller
Submitted to the Graduate Faculty as partial fulfillment of the requirements for the
Doctor of Philosophy Degree in Biomedical Sciences
Dr. Joseph I. Shapiro, Committee Chair
Dr. Christopher J. Cooper, Committee Member
Dr. Deepak Malhotra, Committee Member
Dr. Zijian Xie, Committee Member
Dr. Jiang Liu, Committee member
Dr. Patricia R. Komuniecki, Dean College of Graduate Studies
The University of Toledo August 2012
Copyright 2012, Steven Thomas Haller This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author. An Abstract of Marinobufagenin Induced Uremic Cardiomyopathy: The Role of Passive Immunization, Rapamycin, and CD40 Signaling in The Generation of Renal Fibrosis
By Steven T. Haller Submitted to the Graduate Faculty as partial fulfillment of the requirements for the
Doctor of Philosophy Degree in Biomedical Sciences
The University of Toledo
August 2012
Cardiotonic steroids such as marinobufagenin (MBG) and ouabain are specific ligands for the Na/K-ATPase and represent a relatively new class of steroid hormones.
Uremic cardiomyopathy is characterized by a decrease in diastolic function, left ventricular hypertrophy, oxidant stress, and both cardiac and renal fibrosis. We have shown that MBG, signaling through the Na/K-ATPase, causes many of the adverse pathological effects of experimental uremic cardiomyopathy induced by 5/6th
nephrectomy (PNx) in the rat. The goal of this dissertation is to describe some of the
manipulations we have performed in order to provide potential therapies for the treatment
iii
of uremic cariomyopathy. Specifically, we show that treatment with an anti-MBG antibody drastically reduces cardiac fibrosis in PNx animals. Treatment with rapamycin
(an mTOR inhibitor) produced similar effects with the added benefit of reducing circulating MBG in these animals. In addition, we show that ischemic renal disease is accompanied with elevated levels of the platelet activation marker soluble CD40 ligand
(sCD40L), and its soluble receptor, CD40, may predict outcomes in this disease state.
Data in our PNx model suggests a role for proximal tubular CD40 activation contributing to the development of renal fibrosis, which may be potentiated by cardiotonic steroid signaling through the Na/K-ATPase.
iv
Dedication
To my family who has faithfully stood by my side throughout my academic career. Your undying love and support has inspired me to be a better person. I am forever grateful for the sacrifices you have made for me along the way. Thank you for believing in me. I love you all.
v
Acknowledgments
I am extremely grateful to Drs. Joseph I. Shapiro and Christopher J. Cooper for their
tremendous guidance and faithful support throughout my graduate studies. You have
inspired my passion for translational research. I look forward to working with you in the
future.
I would like to thank my academic advisory committee members: Dr. Zijian Xie, Dr.
Deepak Malhotra, and Dr. Jiang Liu, for there expert advice and guidance throughout my
graduate studies.
Dr. Liu, Thank you for letting me waste away hours in your office discussing my work. I look forward to working with you in the future.
To Dr. Cooper and Holly Burtch for teaching me that being involved in human subjects
research is a privilege, not a right.
To the Clinical Coordinating Center staff, Thank you for supporting and believing in me.
To Pam Brewster, Thank you for your expert advice and support with statistical analysis.
To Carol Woods, Thank you for all of your support.
To Dr. Periyasamy who taught me to walk before I run. I miss you every day.
To Dr. Kennedy, My mentor, my friend, my other brother.
vi
Table of Contents
Abstract iii
Dedication V
Acknowledgments Vi
Table of Contents Vii
Chapter 1 – Literature Review and Introduction 1
Chapter 2 – “Monoclonal antibody against marinobufagenin reverses cardiac fibrosis in
rats with chronic renal failure” (Manuscript) 10
2.1 Abstract 11
2.2 Introduction 12
2.3 Methods 14
2.4 Results 19
2.5 Discussion 20
2.6 Manuscript References 23
2.7 Table and Figure Legends 27 vii
2.8 Table and Figures 29
Chapter 3 – “Rapamycin reduces cardiac fibrosis in experimental uremic cardiomyopathy” (Manusript to be submitted) 33
3.1 Abstract 34
3.2 Introduction 36
3.3 Methods 37
3.4 Results 42
3.5 Discussion 44
3.6 Manuscript References 47
3.7 Table and Figure Legends 52
3.8 Table and Figures 54
Chapter 4 – “Platelet activation in patients with atherosclerotic renal artery stenosis undergoing stent revascularization” (Manuscript) 59
4.1 Abstract 60
4.2 Introduction 61
4.3 Materials and Methods 62
4.4 Results 64
4.5 Discussion 66 viii
4.6 Manuscript References 71
4.7 Table and Figure Legends 78
4.8 Table and Figures 79
Chapter 5 – CD40 mediated fibrosis in chronic and ischemic renal disease 83
5.1 Chronic Kidney Disease and Renal Ischemia 83
5.2 Platelet Activation, CD40 Signaling, and Fibrosis 85
5.3 Preliminary Data: Clinical Trial 86
5.4 Preliminary Data: Animal Studies 90
5.5 Preliminary Data: LLC-PK1 Cells 94
5.6 Conclusions 99
5.7 Chapter 5 References 101
Chapter 6 – Summary and Conclusions 110
ix
Chapter 1-Literature Review and Introduction
1.1 Structure and Function of the Na/K-ATPase
The Na/K-ATPase, originally discovered by Skou (1957), is a member of the P-
type ATPase family, and plays an essential role in regulating the cellular transmembrane
ion gradient by ATP-dependent transport of Na+ and K+ across the plasma membrane.1, 2
In order to maintain ion transport the Na/K-ATPase is in constant flux between two major conformation states, E1 and E2.3 The E1 state has high affinity for Na+ and ATP, while
the E2 state has high affinity for K+.3 The Na/K-ATPase is composed of two
noncovalently linked subunits, α and β which together form the functional unit of the
enzyme.2 There are four α subunit isoforms (α1, α2, α3, and α4), and three β subunit
isoform (β1, β2, and β3) all expressed in a tissue specific mannor.4 The α subunit is the catalytic subunit, which contains specific binding sites for Na+, K+, ATP, and cardiotonic steroids (CTS, ligands of the Na/K-ATPase). The β subunit plays an essential role in regulating the activity of the enzyme.2 A third subunit (γ) contains the conserved FXYD
motif.3 The γ subunit is not considered to be essential for enzymatic function, but has
been proposed to modulate enzymatic activity.5, 6
In addition to the essential ion pumping function, elegant work from the
laboratory of Dr. Xie and collaborators has shown that the cardiotonic steroid ouabain
binds to the α1 subunit of the Na/K-ATPase converting it into a signal transducer capable of activating multiple protein kinase cascades.7-9 Src binds to the Na/K-ATPase α1 1
subunit forming a functional signaling complex 10. CTS bind to the Na/K-ATPase and induce a conformational change which activates Src 10. Src transactivates the epidermal
growth factor receptor (EGFR) which results in the activation of phospholipase C (PLC),
phosphoinsitide 3-kinase (PI3K), mitogen-activated protein kinases (MAPKs), protein kinase C (PKC), and the generation of reactive oxygen species (ROS). 4, 9 Importantly,
ouabain binding to the Na/K-ATPase induces endocytosis of the receptor complex in a
manner consistent with classic receptor tyrosine kinases.11
1.2 Cardiotonic Steroids and Uremic Cardiomyopathy
CTS are specific ligands for the Na/K-ATPase, and have been used to treat heart failure by coupling reduced Na/K-ATPase activity with a reduction in Na+/ Ca2+-
exchanger activity ultimately leading to an accumulation of intracellular sodium and
increases in cytosolic calcium.3, 4 This increase in cytosolic calcium results in increased
cardiac contractile function.4 Endogenous CTS, such as ouabain and marinobufagenin
(MBG), represent a relatively new class of steroid hormones. Endogenous ouabain has
been postulated to be produced from the adrenal cortex and hypothalamus.4 In
amphibians, the biosynthesis of MBG has been proposed to occur via the bile acid
pathway from cholic acids.4 Elevated levels of endogenous CTS have been reported in a
variety of clinical conditions associated with plasma volume expansion such as
congestive heart failure, chronic renal failure, hypertension, renal ischemia, and
preeclamsia.12-19 In animal models, administration of ouabain and MBG have been
shown to cause hypertension, cardiac hypertrophy, and fibrosis.20-22 Furthermore, salt-
loading in Dahl salt-sensitive rats caused an increase in brain derived ouabain, which
2
elevated plasma MBG levels contributing to hypertension in a process mediated by
angiotension II.23
Recent data indicates that chronic kidney disease (CKD) is prevalent, affecting up
to 11% of the US adult population. 24 Platelet activation and inflammation have been
implicated in the progression CKD. 25 Cardiovascular disease is both common and a
major cause of mortality in patients with CKD. 26, 27 This uremic cardiomyopathy is
characterized by a decrease in diastolic function, left ventricular hypertrophy, oxidant
stress, and both cardiac and renal fibrosis. 4, 22, 28 We have shown that MBG, signaling
through the Na/K-ATPase, causes many of the adverse pathological effects of
experimental uremic cardiomyopathy induced by 5/6th nephrectomy (PNx) in the rat. 21
Our group has demonstrated that pharmacologic administration of MBG causes cardiac hypertrophy and fibrosis, as seen in patients, whereas active immunization against MBG attenuated this in PNx. 21, 22 Additionally, cardiac fibroblasts treated with MBG, at
concentrations similar to those reported in experimental and clinical renal failure, has
been shown to stimulate collagen production. 22 This increase in collagen production
appears to be dependent on the Na/K-ATPase-Src-EGFR-ROS signaling cascade. 22 The
transcription factor Friend leukemia integration-1 (Fli-1) has been shown to be a negative regulator of collagen synthesis. 29, 30 PKC- δ phosphorylates Fli-1 and promotes collagen
synthesis. 31 We have shown that MBG signaling through the Na/K-ATPase, caused
PKC- δ translocation to the nucleus leading to Fli-1 phosphorylation and collagen
production. 32 A recent report from our lab has shown that treatment with a monoclonal
antibody directed against MBG (3E9 mAb) in PNx animals resulted in a drastic decrease
in blood pressure, significantly reduced cardiac levels of oxidant stress, increased the 3
expression of Fli-1, and caused a significant reduction in cardiac fibrosis.33
Spironolactone and its major metabolite have been shown to competitively inhibit CTS binding to the Na/K-ATPase.34 We have shown that spironolactone treatment in both
PNx animals and animals receiving MBG infusion attenuated diastolic dysfunction and cardiac fibrosis in these experimental animal models.35 Our findings indicate that spironolactone and the 3E9 monoclonal antibody may offer potential indications for the treatment of uremic cardiomyopathy.
4
References for Literature Review and Introduction
1. Skou JC. The influence of some cations on an adenosine triphosphatase from
peripheral nerves. Biochim Biophys Acta. 1957;23(2):394-401.
2. Kaplan JH. Biochemistry of Na,K-ATPase. Annu Rev Biochem. 2002;71:511-535.
3. Yatime L, Laursen M, Morth JP, Esmann M, Nissen P, Fedosova NU. Structural
insights into the high affinity binding of cardiotonic steroids to the Na+,K+-
ATPase. J Struct Biol.174(2):296-306.
4. Bagrov AY, Shapiro JI, Fedorova OV. Endogenous cardiotonic steroids:
physiology, pharmacology, and novel therapeutic targets. Pharmacol Rev.
2009;61(1):9-38.
5. Arystarkhova E, Wetzel RK, Asinovski NK, Sweadner KJ. The gamma subunit
modulates Na(+) and K(+) affinity of the renal Na,K-ATPase. J Biol Chem.
1999;274(47):33183-33185.
6. Geering K. Function of FXYD proteins, regulators of Na, K-ATPase. J Bioenerg
Biomembr. 2005;37(6):387-392.
7. Xie Z, Askari A. Na(+)/K(+)-ATPase as a signal transducer. Eur J Biochem.
2002;269(10):2434-2439.
8. Xie Z, Cai T. Na+-K+--ATPase-mediated signal transduction: from protein
interaction to cellular function. Mol Interv. 2003;3(3):157-168.
9. Xie Z. Molecular mechanisms of Na/K-ATPase-mediated signal transduction.
Ann N Y Acad Sci. 2003;986:497-503.
5
10. Tian J, Cai T, Yuan Z, Wang H, Liu L, Haas M, Maksimova E, Huang XY, Xie
ZJ. Binding of Src to Na+/K+-ATPase forms a functional signaling complex. Mol
Biol Cell. 2006;17(1):317-326.
11. Liu J, Kesiry R, Periyasamy SM, Malhotra D, Xie Z, Shapiro JI. Ouabain induces
endocytosis of plasmalemmal Na/K-ATPase in LLC-PK1 cells by a clathrin-
dependent mechanism. Kidney Int. 2004;66(1):227-241.
12. Komiyama Y, Dong XH, Nishimura N, Masaki H, Yoshika M, Masuda M,
Takahashi H. A novel endogenous digitalis, telocinobufagin, exhibits elevated
plasma levels in patients with terminal renal failure. Clin Biochem.
2005;38(1):36-45.
13. Rossi G, Manunta P, Hamlyn JM, Pavan E, De Toni R, Semplicini A, Pessina
AC. Immunoreactive endogenous ouabain in primary aldosteronism and essential
hypertension: relationship with plasma renin, aldosterone and blood pressure
levels. J Hypertens. 1995;13(10):1181-1191.
14. Gonick HC, Ding Y, Vaziri ND, Bagrov AY, Fedorova OV. Simultaneous
measurement of marinobufagenin, ouabain, and hypertension-associated protein
in various disease states. Clin Exp Hypertens. 1998;20(5-6):617-627.
15. Fedorova OV, Talan MI, Agalakova NI, Lakatta EG, Bagrov AY. Endogenous
ligand of alpha(1) sodium pump, marinobufagenin, is a novel mediator of sodium
chloride--dependent hypertension. Circulation. 2002;105(9):1122-1127.
16. Fridman AI, Matveev SA, Agalakova NI, Fedorova OV, Lakatta EG, Bagrov AY.
Marinobufagenin, an endogenous ligand of alpha-1 sodium pump, is a marker of
congestive heart failure severity. J Hypertens. 2002;20(6):1189-1194.
6
17. Tian J, Haller S, Periyasamy S, Brewster P, Zhang H, Adlakha S, Fedorova OV,
Xie ZJ, Bagrov AY, Shapiro JI, Cooper CJ. Renal ischemia regulates
marinobufagenin release in humans. Hypertension.56(5):914-919.
18. Lopatin DA, Ailamazian EK, Dmitrieva RI, Shpen VM, Fedorova OV, Doris PA,
Bagrov AY. Circulating bufodienolide and cardenolide sodium pump inhibitors in
preeclampsia. J Hypertens. 1999;17(8):1179-1187.
19. Kolmakova EV, Haller ST, Kennedy DJ, Isachkina AN, Budny GV, Frolova EV,
Piecha G, Nikitina ER, Malhotra D, Fedorova OV, Shapiro JI, Bagrov AY.
Endogenous cardiotonic steroids in chronic renal failure. Nephrol Dial
Transplant.26(9):2912-2919.
20. Kimura K, Manunta P, Hamilton BP, Hamlyn JM. Different effects of in vivo
ouabain and digoxin on renal artery function and blood pressure in the rat.
Hypertens Res. 2000;23 Suppl:S67-76.
21. Kennedy DJ, Vetteth S, Periyasamy SM, Kanj M, Fedorova L, Khouri S, Kahaleh
MB, Xie Z, Malhotra D, Kolodkin NI, Lakatta EG, Fedorova OV, Bagrov AY,
Shapiro JI. Central role for the cardiotonic steroid marinobufagenin in the
pathogenesis of experimental uremic cardiomyopathy. Hypertension.
2006;47(3):488-495.
22. Elkareh J, Kennedy DJ, Yashaswi B, Vetteth S, Shidyak A, Kim EG, Smaili S,
Periyasamy SM, Hariri IM, Fedorova L, Liu J, Wu L, Kahaleh MB, Xie Z,
Malhotra D, Fedorova OV, Kashkin VA, Bagrov AY, Shapiro JI.
Marinobufagenin stimulates fibroblast collagen production and causes fibrosis in
experimental uremic cardiomyopathy. Hypertension. 2007;49(1):215-224.
7
23. Fedorova OV, Agalakova NI, Talan MI, Lakatta EG, Bagrov AY. Brain ouabain
stimulates peripheral marinobufagenin via angiotensin II signalling in NaCl-
loaded Dahl-S rats. J Hypertens. 2005;23(8):1515-1523.
24. Kronenberg F. Emerging risk factors and markers of chronic kidney disease
progression. Nat Rev Nephrol. 2009;5(12):677-689.
25. Tu X, Chen X, Xie Y, Shi S, Wang J, Chen Y, Li J. Anti-inflammatory
renoprotective effect of clopidogrel and irbesartan in chronic renal injury. J Am
Soc Nephrol. 2008;19(1):77-83.
26. Chade AR, Lerman A, Lerman LO. Kidney in early atherosclerosis.
Hypertension. 2005;45(6):1042-1049.
27. Jaradat MI, Molitoris BA. Cardiovascular disease in patients with chronic kidney
disease. Semin Nephrol. 2002;22(6):459-473.
28. Fedorova LV, Raju V, El-Okdi N, Shidyak A, Kennedy DJ, Vetteth S,
Giovannucci DR, Bagrov AY, Fedorova OV, Shapiro JI, Malhotra D. The
cardiotonic steroid hormone marinobufagenin induces renal fibrosis: implication
of epithelial-to-mesenchymal transition. Am J Physiol Renal Physiol.
2009;296(4):F922-934.
29. Czuwara-Ladykowska J, Shirasaki F, Jackers P, Watson DK, Trojanowska M. Fli-
1 inhibits collagen type I production in dermal fibroblasts via an Sp1-dependent
pathway. J Biol Chem. 2001;276(24):20839-20848.
30. Wang Y, Fan PS, Kahaleh B. Association between enhanced type I collagen
expression and epigenetic repression of the FLI1 gene in scleroderma fibroblasts.
Arthritis Rheum. 2006;54(7):2271-2279.
8
31. Jinnin M, Ihn H, Yamane K, Mimura Y, Asano Y, Tamaki K. Alpha2(I) collagen
gene regulation by protein kinase C signaling in human dermal fibroblasts.
Nucleic Acids Res. 2005;33(4):1337-1351.
32. Elkareh J, Periyasamy SM, Shidyak A, Vetteth S, Schroeder J, Raju V, Hariri IM,
El-Okdi N, Gupta S, Fedorova L, Liu J, Fedorova OV, Kahaleh MB, Xie Z,
Malhotra D, Watson DK, Bagrov AY, Shapiro JI. Marinobufagenin induces
increases in procollagen expression in a process involving protein kinase C and
Fli-1: implications for uremic cardiomyopathy. Am J Physiol Renal Physiol.
2009;296(5):F1219-1226.
33. Haller ST, Kennedy DJ, Shidyak A, Budny GV, Malhotra D, Fedorova OV,
Shapiro JI, Bagrov AY. Monoclonal Antibody Against Marinobufagenin Reverses
Cardiac Fibrosis in Rats With Chronic Renal Failure. Am J Hypertens. March
2012 [epub ahead of print].
34. Finotti P, Palatini P. Canrenone as a partial agonist at the digitalis receptor site of
sodium-potassium-activated adenosine triphosphatase. J Pharmacol Exp Ther.
1981;217(3):784-790.
35. Tian J, Shidyak A, Periyasamy SM, Haller S, Taleb M, El-Okdi N, Elkareh J,
Gupta S, Gohara S, Fedorova OV, Cooper CJ, Xie Z, Malhotra D, Bagrov AY,
Shapiro JI. Spironolactone attenuates experimental uremic cardiomyopathy by
antagonizing marinobufagenin. Hypertension. 2009;54(6):1313-1320.
9
Chapter 2 – Manuscript
Title: Monoclonal Antibody Against Marinobufagenin Reverses Cardiac Fibrosis in Rats With Chronic Renal Failure
Authors:
Steven T. Haller1, David J. Kennedy1,2, Amjad Shidyak2, George V. Budny1, Deepak Malhotra1,Olga V. Fedorova3, Joseph I. Shapiro1 and Alexei Y. Bagrov3
1College of Medicine, University of Toledo, Toledo, Ohio, USA;
2Departmentof Cell Biology, Cleveland Clinic Lerner Research Institute, Cleveland,
Ohio,USA; 3Laboratory of Cardiovascular Science, National Institute on Aging,
NIH,Baltimore, Maryland, USA.
Corresponding Author:
Dr. Alexei Y. Bagrov at the National Institute on Aging, NIH, 5600 Nathan Shock Drive,
Baltimore, MD 21224
Published in American Journal of Hypertension. March 2012 1. doi:10.1038/ajh.2012.17.
[Epub ahead of print]
10
2.1 Abstract
Cardiotonic steroids (CTS) are implicated in pathophysiology of uremic cardiomyopathy. In the present study, we tested whether a monoclonal antibody (mAb) against the bufadienolide CTS, marinobufagenin (MBG), alleviates cardiac hypertrophy and fibrosis in partially nephrectomized (PNx) rats. In PNx rats, we compared the effects of 3E9 anti-MBG mAb and of Digibind, an affinity-purified digoxin antibody, on blood pressure and cardiac hypertrophy and fibrosis following 4 weeks after the surgery. In
PNx rats, a fourfold elevation in plasma MBG levels was associated with hypertension, increased cardiac levels of carbonylated protein, cardiac hypertrophy, a reduction in cardiac expression of a nuclear transcription factor which is a negative regulator of collagen synthesis, Friend leukemia integration-1 (Fli-1), and an increase in the levels of collagen-1. A single intraperitoneal administration of 3E9 mAb to PNx rats reduced blood pressure by 59 mm Hg for 7 days and produced a significant reduction in cardiac weight and cardiac levels of oxidative stress, an increase in the expression of Fli-1, and a reduction in cardiac fibrosis. The effects of Digibind were similar to those of 3E9 mAb, but were less pronounced. In experimental chronic renal failure, elevated levels of MBG contribute to hypertension and induce cardiac fibrosis via suppression of Fli-1, representing a potential target for therapy.
11 2.2 Introduction
Uremic cardiomyopathy is a major cause of morbidity and mortality in patients
with chronic kidney disease.1 Despite considerable recent progress in the understanding
of the pathogenesis of uremic cardiomyopathy, there is clearly a niche for novel
approaches to its treatment.1,2 An increasing body of evidence indicates that one of the
factors implicated in pathogenesis of uremic cardiomyopathy is the group of hormones
known as endogenous cardiotonic steroids (CTS).3 CTS regulate sodium pump activity
at a cellular level and are implicated in the regulation of natriuresis and vascular tone.3
Many of the effects of these hormones appear to derive from a signaling function of the
Na/K-ATPase; in particular, this signaling stimulated by CTS leads to cardiac
hypertrophy and fibrosis.4,5
Previously, we demonstrated that circulating concentrations of marinobufagenin
(MBG) (14,15β-Epoxy-3β,5-dihydroxy-5β-bufa-20,22 dienolide), an endogenous bufadienolide CTS, are elevated in patients with renal failure and in partially nephrectomized rats (PNx).5,6 In PNx rats, also we observed increased cardiac and
plasma levels of carbonylated proteins as well as other evidence for signaling through the
Na/K-ATPase such as activation of Src and mitogen-activated protein kinase (MAPK).5,6
In these studies, active immunization of PNx rats against MBG dramatically reduced cardiac hypertrophy and fibrosis and systemic oxidant stress, as well as evidence of
Na/K-ATPase signaling. Conversely, chronic administration of MBG to normotensive rats to achieve plasma concentrations of MBG as seen with PNx, produced cardiac phenotype similar to PNx.5,6
12 The transcription factor, Friend leukemia integration-1 (Fli-1), a member of the
ETS family, is a negative regulator of collagen synthesis,7 and reduced levels of Fli-1
were documented in skin fibroblasts of patients with scleroderma.8,9 Recent evidence
indicates that suppression of Fli-1 is also implicated in profibrotic signaling by CTS. In
vitro, we have demonstrated that nanomolar concentrations of MBG stimulate collagen
production by dermal, cardiac, and renal fibroblasts by a mechanism involving protein
kinase C δ-dependent phosphorylation and depletion of Fli-1.7 Interestingly, when we
stably transfected renal fibroblasts with a Fli-1 expression vector which dramatically increased Fli-1 expression, the basal expression of procollagen was decreased and MBG treatment did not increase procollagen expression or appreciably reduce Fli-1
expression.7
Recently, we developed two anti-MBG monoclonal antibodies (mAb), 3E9 and
4G4.10 In our previous experiments, 3E9 mAb exceeded 4G4 with respect to reversal of
MBG-induced Na/K ATPase inhibition, and potently reduced blood pressure and restored
vascular sodium pump activity in hypertensive Dahl-S rats and in pregnant Sprague-
Dawley rats rendered hypertensive by NaCl supplementation. Because of these
properties, in the present experiment we used 3E9 mAb for in vivo MBG
immunoneutralization, while 4G4 mAb which exhibits high affinity to MBG in
competitive immunoassays was chosen for MBG measurement.10 In the present
experiment, in PNx rats, we studied effects of 3E9 anti-MBG mAb on arterial pressure,
cardiac fibrosis and oxidative stress, and cardiac expression of Fli-1. We also compared
effects of 3E9 mAb to those of Digibind (the Fab fragments of ovine digoxin antibody)
which has been demonstrated to both bind endogenous CTS,11 as well as lower blood
13 pressure in patients with preeclampsia,12,13 a clinical syndrome known to have elevated
CTS levels.10,14
2.3 Methods
2.3.1 Animal studies
All animal experimentation described in this article was conducted in accordance
with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory
Animals under protocols approved by the University of Toledo Institutional Animal Care
and Use Committee. Male Sprague-Dawley rats (250–300 g) were used for these studies.
Eight sham nephrectomized rats comprised the control group. In 18 rats, PNx (5/6
nephrectomy) was produced by surgical removal of the right kidney and ligation of the
two-thirds of the arterial supply to the left kidney as reported previously in detail.15 In
brief, rats were anesthetized with a mixture of 100% oxygen and 5% isoflurane, an
incision was made in the left flank, through which the left kidney was pulled out, and
arteries supplying to upper and lower poles were ligated. After a week, the right kidney
was decapsulated to avoid removal of adrenal gland, artery, vein, and ureter were ligated,
and the kidney was removed. T his maneuver produces sustained hypertension within 2
weeks.5,6 At 4 weeks following PNx, these rats were intraperitoneally administered
vehicle (n = 6), Digibind (n = 6) or 3E9 anti-MBG mAb (n = 6). The dose of Digibind
(10 μg/kg) was similar to that previously administered to patients with preeclampsia,12,13
and the dose of 3E9 mAb (50 μg/kg) was the same as that previously reported to reverse
the EC75 to the inhibition of the Na/K-ATPase by MBG in rat renal outer medulla in
vitro, and to reduce blood pressure in hypertensive Dahl-S rats in vivo.10 Blood pressure
14 was determined using the tail cuff method by IITC (Amplifier model 229, Monitor model
31, Test chamber Model 306; IITC Life Science, Woodland Hills, CA) at baseline, 3, 24,
48 h and at 1 week following antibody treatment (5 weeks after PNx). Then rats were killed and the heart weight and cardiac histology were determined. Plasma samples were stored at −80 °C for determination of CTS.
2.3.2 Oxidative stress markers
Levels of oxidative stress were assessed by measurement of protein carbonyl levels and determination of intracellular production of reactive oxygen species using the fluorescent probe dye dihydroethidium (DHE). Total protein carbonyl concentration of the plasma and left ventricular homogenate was determined by enzyme-linked immunosorbent assay using the BIOCELL PC Test kit (Northwest Life Science
Specialties, Vancouver, WA). Production of reactive oxygen species was detected by
DHE (Invitrogen Molecular Probes, Eugene, OR) as described previously.16,17 Briefly, left ventricle tissue was frozen in optimal cutting temperature compound, and transverse sections (10 μm) were generated with a cryostat and placed on glass slides. Tissue sections were incubated with 5 μmol/l DHE at 37 °C for 20 min according to the manufacturer’s instructions. Red fluorescence was assessed by using an Olympus
FSX100 box type fluorescence imaging device (Olympus America, Center Valley, PA).
The excitation wavelength was 488 nm with emission at 585 nm. Fluorescence intensity was analyzed by the use of Image J (version 1.32j) software (National Institutes of
Health, USA; http://rsb.info.nih.gov/ij/).
15 2.3.3 Creatinine and creatinine clearance
At the conclusion of the study, 24 h urine samples were collected. At the end of
urine collection, animals were killed and blood samples were obtained from abdominal
aorta. Plasma creatinine was measured with a colorimetric method using a commercial kit from Teco Diagnostics (Anaheim, CA, cat. no. C515-480). Creatinine standards or plasma samples were mixed with the picric acid reagent and creatinine buffer reagent provided with the kit. The optical density value at 510 nm was measured immediately after and at 15 min. The differences between the two time points were used to calculate the creatinine concentrations. Creatinine clearance was calculated using the following formula: (urine Cr × urine Vol (ml)/plasma Cr × 24 h × 60 min).
2.3.4 Western blot analyses of Fli-1 and collagen-1
Western Blot analysis was performed on proteins from tissue homogenates as previously reported.7 The left ventricles from the heart were homogenized in ice-cold
RIPA lysis buffer (pH 7.0) Santa Cruz Biotechnology (Santa Cruz, CA; sc-24948). The
homogenate was centrifugated at 1,400g for 30 s at 4 °C. The supernatant was discarded
and the pellet fraction was resuspended in 5% sodium dodecyl sulfate (SDS) and 50
mmol/l Tris-HCl (pH 7.4). The protein was quantified in the resuspended pellet fraction
and was solubilized in sample buffer (2% SDS, 5% β-mercaptoethanol, 20% glycerol,
0.005% bromophenol blue, and 50mmol/l Tris- HCl; pH 7.0). The proteins, obtained
from tissue homogenates, were resolved on an SDS-polyacrylamide gel electrophoresis
(PAGE) using Precast Ready Gels 4–15% Tris-HCl, purchased from Bio-Rad (Hercules,
CA). Ten microgram of protein per sample were loaded into each well. The proteins
16 from the gel were electrotransferred to a nitrocellulose membrane. The membrane was
blocked with 5% nonfat dry milk in 20 mmol/l Tris-HCl (pH 7.5, 150 mmol/l NaCl, and
0.1% Tween 20). Goat anti-type 1 collagen antibody (Southern Biotech, Birmingham,
AL) was used to probe for collagen-1, and secondary anti-goat antibody was purchased
from Santa Cruz Biotechnology. To probe for Fli-1, we used rabbit polyclonal anti-
Fli1(C19) antibody (Santa Cruz Biotechnology; 1:500) and peroxidase-conjugated anti-
rabbit antiserum (Amersham, Piscataway, NJ; 1:1,000). For detection, we used ECL and
ECL plus purchased from Amersham Biosciences. Loading conditions were controlled using anti-actin mouse monoclonal antibody (Santa Cruz Biotechnology).
2.3.5 Histology
Trichrome staining was performed on left ventricular tissues and tissue fibrosis was quantified as previously reported.6–7,15 Left ventricle sections were immediately
fixed in 4% formalin buffer solution (pH 7.2) for 18 h, dehydrated in 70% ethanol, and
then embedded in paraffin and cut with a microtome. Trichrome staining was then
performed and fibrosis was quantified using ImageJ software. For quantitative
morphometric analysis, five random sections of trichrome slides were electronically
scanned into an RGB image which was subsequently analyzed using Image J (version
1.32j) software. The amount of fibrosis was then estimated from the RGB images with a
macro written by the authors (J.I.S.) by converting pixels of the image with substantially
greater (>120%) blue than red intensity to have the new, gray scale amplitude = 1,
leaving other pixels as with amplitude = 0.
17 2.3.6 MBG immunoassay
For measurement of MBG, plasma samples were extracted using C18 SepPak
cartridges (Waters, Cambridge, MA). Cartridges were activated with 10 ml acetonitrile and washed with 10 ml water. Then 0.5 ml plasma samples were applied to the cartridges and consecutively eluted in the same vial with 7 ml 20% acetonitrile followed by 7 ml
80% acetonitrile and vacuum dried. Before immunoassays, samples were reconstituted in the initial volume of assay buffer. MBG was measured using a fluoroimmunoassay based on a murine anti-MBG 4G4 mAb recently described in detail.10 This assay is based on competition between immobilized antigen (MBG-glycoside-thyroglobulin) and MBG or other cross-reactants, within the sample for a limited number of binding sites on 4G4 anti-MBG mAbs. Secondary (goat anti-mouse) antibody labeled with nonradioactive europium was obtained from Perkin-Elmer (Waltham, MA). Data on cross-reactivity of the 4G4 mAb used for determination of MBG levels and of 3E9 mAb used for in vivo administration was reported previously.10 MBG (>98% high performance liquid
chromatography pure) was purified from secretions from parotid glands of Bufo marinus
toads as reported previously.10
2.3.7 Statistical analyses
The results are presented as means ± s.e.m. Data were analyzed using one way analysis of variance followed by Newman–Keuls test (intragroup analyses), by repeated measures analysis of variance followed by Newman–Keuls test (intergroup analyses), and by two-tailed t-test (when applicable) (GraphPad Prism software, San Diego, CA). A two-sided P value of less than 0.05 was considered to be statistically significant.
18 2.4 Results
In Sprague-Dawley rats, PNx led to hypertension, marked increases in plasma creatinine and oxidative stress as assessed by plasma levels of carbonylated protein
(Table 2.1). Plasma levels of MBG in PNx rats were elevated fourfold vs. that in sham operated animals (Figure 2-1a). Rats subjected to PNx treated with vehicle developed cardiac hypertrophy (Figure 2-2a) which was accompanied by activation of cardiac oxidative stress assessed by carbonylated protein and DHE staining (Figure 2-2b, c).
Development of renal failure in rats was also associated with cardiac fibrosis assessed by computer-assisted morphological analysis and increased levels of collagen-1 in left ventricular myocardium (Figure 2-3) as we have previously reported.5,6 Cardiac levels of
Fli-1 in PNx rats were markedly reduced vs. that in sham-operated animals (Figure 2-3a).
Figures 2-1 and 2-2 summarize data on the effect of administration of 3E9 anti-
MBG mAb and Digibind on blood pressure, heart weight, and cardiac levels of carbonylated protein. A single administration of Digibind produced a transient decrease in arterial pressure while, in contrast, administration of 3E9 mAb resulted in a substantial and sustained decrease in systolic blood pressure following 1 week of antibody administration (Figure 2-1d). In addition to depressor effect, administration of 3E9 mAb and Digibind to PNx rats was associated with reduction in the serum levels of creatinine and a concomitant increase in creatinine clearance (Figure 2-1b, c).
Administration of both antibodies was associated with a reduction in cardiac weight and a decrease in the cardiac expression of carbonylated protein (Figure 2-2), as well as increase in the left ventricular expression of Fli-1 protein (Figure 2-3a) along with reductions in cardiac collagen 1 protein expression and morphological evidence of
19 fibrosis (Figure 2-3b, c). These effects of 3E9 mAb were more pronounced as compared
to those of Digibind.
2.5 Discussion
The main observation of the present experiment is that a single administration of a
mAb against an endogenous Na/KATPase inhibitor, MBG, to rats with experimental
renal failure produced a sustained depressor effect associated with a dramatic reduction in
cardiac fibrosis and increase in cardiac levels of Fli-1, a negative regulator of collagen
synthesis. Fli-1 belongs to a family of Ets oncogenes, and it competes with another
transcription factor, ETS-1, to maintain a balance between stimulation and repression of
Col1a2 gene promoter.8 Fli-1 is implicated in dermal fibrosis and it exhibits direct effect
on collagen-1 synthesis in dermal fibroblasts,8 and we have observed that decreases in
Fli-1 expression appear to be necessary for CTS to stimulate fibroblast collagen
production.7
Our present results demonstrate that blockade of the CTS-Na/ K-ATPase signal cascade can actually reverse established cardiac fibrosis in the PNx model. Our previous studies demonstrated that cardiac fibrosis was well established after 4 weeks following
PNx in the Sprague-Dawley rat.5,6 In the current study, treatment was administered to
rats at 4 weeks following PNx, and animals were killed 1 week later. While the vehicle
treatment group, studied 5 weeks after PNx, demonstrated similar degrees of cardiac
hypertrophy and fibrosis to what we had previously reported at 4 weeks,5,6 both Digibind
and 3E9 treated animals had remarkable clearing of myocardial fibrosis over the next
week accompanied by upregulation of cardiac Fli-1. These data further indicate that
20 MBG-dependent Fli-1 downregulation is implicated in the pathogenesis of cardiac
fibrosis seen with experimental chronic renal failure. Our present observations that
immunoneutralization of MBG was accompanied by reduction in systemic and cardiac
levels of oxidative stress agrees with previous data demonstrating that generation of
reactive oxygen species is implicated in MBG-dependent cell signaling.18,19
Previous studies in PNx rats implemented remnant kidney fibrosis in the
progression of renal failure in this model.20,21 Although absence of renal morphology
data is a limitation of the present study, we found that in PNx rats immunoneutralization
of CTS with both 3E9 mAb and Digibind reduced plasma creatinine concentration and
produced a substantial increase in creatinine clearance. This observation suggests that in
PNx rats beneficial effects of MBG immunoneutralization are not limited to pressor and
cardiac effects, and warrants further studies of the role of CTS in the pathogenesis of
renal fibrosis.
Notably, in the present study, in the case of Digibind, reduction of cardiac fibrosis
occurred in the absence of a sustained blood pressure-lowering effect. Thus, in the present experiment, Digibind and 3E9 mAb in PNx rats exhibited comparable antifibrotic effects in the presence of markedly varying effects on the blood pressure within 1 week after a single injection; while the blood pressure-lowering effect of anti-MBG mAb was profound and sustained, the depressor effect of Digibind was minor and transient. We, therefore, propose that in the present study, both antibodies exhibited blood pressure independent antifibrotic effects, which agrees with our previous data demonstrating that a pronounced antifibrotic effect of active immunization of PNx rats against MBG was associated with a very minor effect on the blood pressure.5,6
21 The 3E9 anti-MBG mAb employed in the current study is highly selective for
bufadienolide CTS and does not cross react with cardenolide sodium pump inhibitors and
other steroid hormones.10 Thus, in a competitive immunoassay, the 3E9 mAb exhibited
substantial cross-reactivity only with two bufadienolides, telocinobufagin, a possible precursor of MBG which was reported to be elevated in plasma of patients with uremia,22
and cinobufotalin which differs from MBG in having one extra hydroxyl group.10
Previously, we reported that following high-performance liquid chromatography-
fractionation of CTS from preeclamptic placentae, a competitive immunoassay based on
Digibind exhibited reactivity to high-performance liquid chromatography fractions having retention times similar to that seen with MBG and other bufadienolides, but not to ouabain-like immunoreactive material.23 Most recently, in patients with chronic kidney disease and in PNx rats we demonstrated that increase in plasma CTS detected by
Digibind is likely to reflect an increase in the levels of MBG.24 These observations
suggest that in renal failure MBG represents a target for Digibind.
In conclusion, in experimental chronic renal failure, elevated levels of MBG
contribute to hypertension and induce cardiac fibrosis via suppression of Fli-1,
representing a potential target for therapy. The effectiveness of 3E9 mAb for reversing
the cardiac disease in PNx animals and the fact that 3E9 mAb exhibits a long-lasting effect following a single injection, suggests a potential role for MBG immunoneutralization in patients with uremic cardiomyopathy.
22
2.6 Manuscript References
1. London GM. Cardiovascular disease in chronic renal failure: pathophysiologic
aspects. Semin Dial 2003; 16:85–94.
2. Remppis A, Ritz E. Cardiac problems in the dialysis patient: beyond coronary
disease. Semin Dial 2008; 21:319–325.
3. Bagrov AY, Shapiro JI. Endogenous digitalis: pathophysiologic roles and
therapeutic applications. Nat Clin Pract Nephrol 2008; 4:378–392.
4. Kometiani P, Li J, Gnudi L, Kahn BB, Askari A, Xie Z. Multiple signal transduction
pathways link Na+/K+-ATPase to growth-related genes in cardiac myocytes.
The roles of Ras and mitogen-activated protein kinases. J Biol Chem 1998;
273:15249–15256.
5. Kennedy DJ, Vetteth S, Periyasamy SM, Kanj M, Fedorova L, Khouri S,
Kahaleh MB, Xie Z, Malhotra D, Kolodkin NI, Lakatta EG, Fedorova OV,
Bagrov AY, Shapiro JI. Central role for the cardiotonic steroid marinobufagenin
in the pathogenesis of experimental uremic cardiomyopathy. Hypertension
2006; 47:488–495.
6. Elkareh J, Kennedy DJ, Yashaswi B, Vetteth S, Shidyak A, Kim EG, Smaili S,
Periyasamy SM, Hariri IM, Fedorova L, Liu J, Wu L, Kahaleh MB, Xie Z, Malhotra
D, Fedorova L, Kashkin VA, Bagrov AY, Shapiro JI. Marinobufagenin stimulates
fibroblast collagen production and causes fibrosis in experimental uremic
cardiomyopathy. Hypertension 2007;49:215-224.
23 7. Elkareh J, Periyasamy SM, Shidyak A, Vetteth S, Schroeder J, Raju V, Hariri IM,
El-Okdi N, Gupta S, Fedorova L, Liu J, Fedorova OV, Kahaleh MB, Xie Z, Malhotra
D, Watson DK, Bagrov AY, Shapiro JI. Marinobufagenin induces increases in
procollagen expression in a process involving protein kinase C and Fli-1:
implications for uremic cardiomyopathy. Am J Physiol Renal Physiol 2009;
296:F1219–F1226.
8. Czuwara-Ladykowska J, Shirasaki F, Jackers P, Watson DK, Trojanowska M. Fli-1
inhibits collagen type I production in dermal fibroblasts via an Sp1-dependent
pathway. J Biol Chem 2001; 276:20839–20848.
9. Wang Y, Fan PS, Kahaleh B. Association between enhanced type I collagen
expression and epigenetic repression of the FLI1 gene in scleroderma fibroblasts.
Arthritis Rheum 2006; 54:2271–2279.
10. Fedorova OV, Simbirtsev AS, Kolodkin NI, Kotov AY, Agalakova NI, Kashkin
VA, Tapilskaya NI, Bzhelyansky A, Reznik VA, Frolova EV, Nikitina ER, Budny
GV, Longo DL, Lakatta EG, Bagrov AY. Monoclonal antibody to an endogenous
bufadienolide, marinobufagenin, reverses preeclampsia-induced Na/K-ATPase
inhibition and lowers blood pressure in NaCl-sensitive hypertension. J Hypertens
2008; 26:2414–2425.
11. Fedorova OV, Tapilskaya NI, Bzhelyansky AM, Frolova EV, Nikitina ER,
Reznik VA, Kashkin VA, Bagrov AY. Interaction of Digibind with endogenous
cardiotonic steroids from preeclamptic placentae. J Hypertens 2010; 28:361–366.
24 12. Goodlin RC. Antidigoxin antibodies in eclampsia. N Engl J Med 1988; 318:518–
519.
13. Adair CD, Buckalew V, Taylor K, Ernest JM, Frye AH, Evans C, Veille JC.
Elevated endoxin-like factor complicating a multifetal second trimester pregnancy:
treatment with digoxin-binding immunoglobulin. Am J Nephrol 1996; 16:
529–531.
14. Lopatin DA, Ailamazian EK, Dmitrieva RI, Shpen VM, Fedorova OV, Doris PA,
Bagrov AY. Circulating bufodienolide and cardenolide sodium pump inhibitors in
preeclampsia. J Hypertens 1999; 17:1179–1187.
15. Shapiro JI, Harris DC, Schrier RW, Chan L. Attenuation of hypermetabolism in the
remnant kidney by dietary phosphate restriction in the rat. Am J Physiol 1990;
258:F183–F188.
16. Zanetti M, d’Uscio LV, Peterson TE, Katusic ZS, O’Brien T. Analysis of superoxide
anion production in tissue. Methods Mol Med 2005; 108:65–72.
17. Takimoto E, Champion HC, Li M, Ren S, Rodriguez ER, Tavazzi B, Lazzarino G,
Paolocci N, Gabrielson KL, Wang Y, Kass DA. Oxidant stress from nitric oxide
synthase-3 uncoupling stimulates cardiac pathologic remodeling from chronic
pressure load. J Clin Invest 2005; 115:1221–1231.
18. Xie Z, Kometiani P, Liu J, Li J, Shapiro JI, Askari A. Intracellular reactive oxygen
species mediate the linkage of Na+/K+-ATPase to hypertrophy and its marker
genes in cardiac myocytes. J Biol Chem 1999; 274:19323–19328.
25 19. Priyadarshi S, Valentine B, Han C, Fedorova OV, Bagrov AY, Liu J, Periyasamy
SM, Kennedy D, Malhotra D, Xie Z, Shapiro JI. Effect of green tea extract on
cardiachypertrophy following 5/6 nephrectomy in the rat. Kidney Int 2003; 63:1785–
1790.
20. An WS, Kim HJ, Cho KH, Vaziri ND. Omega-3 fatty acid supplementation
attenuates oxidative stress, inflammation, and tubulointerstitial fibrosis in
the remnant kidney. Am J Physiol Renal Physiol 2009; 297:
F895–F903.
21. Sun L, Zhang D, Liu F, Xiang X, Ling G, Xiao L, Liu Y, Zhu X, Zhan M, Yang Y,
Kondeti VK, Kanwar YS. Low-dose paclitaxel ameliorates fibrosis in the remnant
kidney model by down-regulating miR-192. J Pathol 2011; 225:364–377.
22. Komiyama Y, Dong XH, Nishimura N, Masaki H, Yoshika M, Masuda M,
Takahashi H. A novel endogenous digitalis, telocinobufagin, exhibits elevated plasma
levels in patients with terminal renal failure. Clin Biochem 2005; 38:36–45.
23. Fedorova OV, Tapilskaya NI, Bzhelyansky AM, Frolova EV, Nikitina ER,
Reznik VA, Kashkin VA, Bagrov AY. Interaction of Digibind with endogenous
cardiotonic steroids from preeclamptic placentae. J Hypertens 2010; 28:361–366.
24. Kolmakova EV, Haller ST, Kennedy DJ, Isachkina AN, Budny GV, Frolova EV,
Piecha G, Nikitina ER, Malhotra D, Fedorova OV, Shapiro JI, Bagrov AY.
Endogenous cardiotonic steroids in chronic renal failure. Nephrol Dial Transplant
2011; 26:2912–2919.
26 2.7 Table and Figure Legends
Table 2.1. Physiological measurements in control and PNx rats. Means ± SEM. *-
P<0.01 vs.control group. Two-tailed t-test or Wilcoxon test (plasma carbonylated protein). PNx, partially nephrectomized rats. *P<0.01 vs. control group.
Figure 2-1. Plasma levels of MBG (A), creatinine (B), and creatinine clearance (C)
in sham-operated (Sham) and PNx rats, treated with vehicle (Veh), Digibind (DG)
or anti-MBG mAb (3E9). Effects of administration of 3E9 anti-MBG mAb and of
Digibind to PNx rats on systolic BP (D). Means ± SEM from 6 observations. A: (*)
- P<0.01 vs. Sham by two-tailed t-test. B and C: (*) - P<0.05, (**) - P<0.001 vs.
Sham, (#) - P<0.05, (##) - P<0.01 vs. vehicle by one-way ANOVA followed by
Newman-Keuls test. D: By repeated measures ANOVA and Newman-Keuls test:
Digibind vs. vehicle – P<0.05; 3E9 mAb vs. vehicle – P<0.01; Digibind vs. 3E9 –
P<0.01.
Figure 2-2. Effects of administration of 3E9 anti-MBG mAb and of Digibind to PNx
rats on heart weight (A), and on cardiac levels of oxidative stress assessed by
measurement of carbonylated protein (B) and DHE fluorescence intensity (C);
upper panels, representative measurements; lower panel – quantitative
measurements, mean±SEM of 4 densiometry determinations). PNx – partially
nephrectomized rats. Vehicle – PNx rats administered vehicle. Means ± SEM
from 6 observations. A-C: By one-way ANOVA and Newman-Keuls test (*) –
27 P<0.05 and (**) – P<0.01 vs. Sham. (#) – P<0.05 and (##) – P<0.01 vs. vehicle treated
PNx rats.
Figure 2-3. Representative (upper panel) and quantitative (lower panel, mean±SEM of 4 densiometry measurements) analysis of Fli-1 (A) and collagen-1
(B) Western blots performed on cardiac tissues from the different groups. Actin
was used to control loading. C – representative (upper panel) and quantitative
(lower panel, mean±SEM of 4 densiometry measurements) trichrome-stained
photomicrographs obtained from cardiac tissue derived from the different
experimental groups. Sham – sham-operated rats, PNx – partially
nephrectomized rats, Veh – PNx rats administered vehicle, DG – PNx rats
administered Digibind, 3E9 – PNx rats administered 3E9 anti-MBG mAb. By one-way
ANOVA and Newman-Keuls test: (*) – P<0.01 vs. Sham; (#) – P<0.05, (##)
– P<0.01 vs. Veh.
28 2.8 Table and Figures
Table 2.1
29 Figure 2-1.
30 Figure 2-2.
31 Figure 2-3.
32 Chapter 3 – Manuscript
Title:
Rapamycin Reduces Cardiac Fibrosis in Experimental Uremic Cardiomyopathy
Authors:
Steven T. Haller1, George V. Budney1, Joe Xie1, Jiang Tian1, Mohamed Taleb1, Deepak Malhotra1, Olga V. Fedorova2, Alexei Y. Bagrov2, and Joseph I. Shapiro1
1University of Toledo Collage of Medicine, Toledo, Ohio
2 Laboratory of Cardiovascular Science, National Institute on Aging, Baltimore, Md.
Corresponding Author:
Dr. Joseph I. Shapiro, University of Toledo, Collage of Medicine, 3000 Arlington Ave., Toledo, OH
To be Submitted to Hypertension
33 3.1 Abstract
Background: We have shown that experimental uremic cardiomyopathy causes
cardiac fibrosis and is associated with increased levels of the cardiotonic steroid
marinobufagenin (MBG), an inhibitor of the Na/K-ATPase. The mammalian target of
rapamycin (mTOR) is a serine/threonine kinase implicated in the progression of many
different forms of renal disease. Treatment with rapamycin (an mTOR inhibor) has been
shown to attenuate inflammation, and renal fibrosis in experimental models of renal
disease. The use of rapamycin in the setting of experimental uremic cardiomyopathy has
not been defined.
Materials and Methods: Male Sprague Dawley rats weighing between 250-300
gms were used for these studies. Rats were divided into six groups. In the first group,
partial nephrectomy (PNx) was performed as we have previously described. This
maneuver produces sustained hypertension by 2 weeks under these conditions. In the
second group, PNx was performed and rapamycin was administered (0.2mg/kg/day). The
third group received both rapamycin (0.2mg/kg/day) and MBG (10µg/kg/day). The
fourth and fifth groups were administered MBG alone and rapamycin alone. The sixth
group consisted of sham operated controls. All treatments were performed for 4 weeks
with the use of osmotic minipumps.
Results: The PNx animals showed an extensive increase in plasma MBG levels,
systolic BP, and cardiac fibrosis. Plasma MBG levels were significantly decreased in the
PNx-rapamycin animals compared to PNx (124 ± 15 vs 342 ± 20, P<0.01). The PNx- rapamycin animals showed a substantial decrease in cardiac fibrosis compared to PNx
34
animals. MBG treated animals had significant increases in systolic BP, and cardiac fibrosis compared to controls. Rapamycin treatment in combination with MBG did not significantly attenuate these effects.
Conclusion: The mTOR pathway has been implicated in the generation of renal fibrosis during renal failure. Our results suggest that the mTOR pathway may have a significant impact in the generation of cardiac fibrosis. Treatment with rapamycin may provide a novel therapy for reducing cardiac fibrosis in the setting of uremic cardiomyopathy.
35
3.2 Introduction
The high mortality rate in patients with chronic renal failure is ultimately due to
severe cardiovascular disease. 1 This uremic cardiomyopathy is characterized by cardiac
hypertrophy, diastolic dysfunction, and cardiac fibrosis along with elevated circulating
concentrations of the cardiotonic steroid marinobufagenin (MBG), a ligand of the Na/K-
ATPase. MBG belongs to a family of bufadienolides previously described in
amphibians. 2 In toads, the biosynthesis of MBG occurs via the bile acid pathway from
cholic acids. 2 This pathway may also be responsible for the production of MBG in
mammals. We have shown that MBG is elevated in patients with renal failure 3 and in
rats subjected to partial nephrectomy (PNx), and those with pharmacologic administration of MBG developed a similar cardiomyopathy as seen in patients, whereas active immunization against MBG attenuated this in PNx. 4, 5 In PNx rats, we also
observed increased cardiac and plasma levels of carbonylated proteins as well as
evidence for signaling through the Na/K-ATPase such as activation of Src and mitogen-
activated protein kinase (MAPK).4, 5 A recent report from our group has shown that
treatment with a monoclonal antibody directed against MBG drastically reduced cardiac
fibrosis in PNx animals.6
The mammalian target of rapamycin (mTOR) is a serine/threonine kinase
composed of two signaling complexes, mTORC1 and mTORC2. 7 The mTORC1
complex is involved in cellular proliferation and growth, while mTORC2 is involved the
regulation of the cytoskeleton. 8 The mTOR pathway has been implicated in the
progression of many different forms of renal disease including experimentally induced
uremic cardiomyopathy .9, 10 Treatment with rapamycin (an mTORC1 inhibitor) has been 36
shown to attenuate inflammation, fibrosis, and cardiac hypertrophy in experimental
models of renal disease. 9, 10 Rapamycin is also a competitive inhibitor of CYP27A1, a
key rate limiting enzyme of the bile acid pathway .11
Based on this background, the primary goals of the present study were to
determine the effects of rapamycin on cardiac fibrosis and MBG production using the rat
PNx model of uremic cardiomyopathy.
3.3 Methods
3.3.1 Animal Studies
All animal experimentation described in this article was conducted in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory
Animals under protocols approved by the University of Toledo Institutional Animal Care and Use Committee. Male Sprague-Dawley rats (250–300 g) were used for these studies.
Rats were divided into six groups. In the first group, partial nephrectomy (PNx) was performed as we have previously described.5 In the second group, PNx was performed
and rapamycin (RAPA) was administered (0.2mg/kg/day). 12 The third group received
both rapamycin (0.2mg/kg/day) and MBG (10µg/kg/day). The fourth and fifth groups
were administered MBG alone (10µg/kg/day) and rapamycin alone (0.2mg/kg/day). The
dose of MBG is the same as we have previously reported to induce the physiological
changes associate with uremic cardiomyopathy .4, 5 The sixth group consisted of sham
37
operated controls. All treatments were performed for 4 weeks with the use of osmotic
minipumps (Alzet, model 2004). Minipumps were inserted SC through a flank incision.
3.3.2 Blood Pressure, Cardiac Physiology, and Other In Vivo Measurements
Blood pressure was determined using the tail cuff method by IITC (Amplifier
model 229, Monitor model 31, Test chamber Model 306; IITC Life Science, Woodland
Hills, CA) at baseline, and once weekly for four weeks. Then rats were euthanized and
the heart weight and cardiac histology were determined. Plasma samples were stored at
−80 °C for biochemical analysis. Plasma MBG and creatinine were measured as we have
previously described. 6, 13
3.3.3 Oxidative stress markers
Levels of oxidative stress were assessed by determination of intracellular production of reactive oxygen species using the fluorescent probe dye dihydroethidium
(DHE). Production of reactive oxygen species was detected by DHE (Invitrogen
Molecular Probes, Eugene, OR) as described previously. 14, 15 Briefly, left ventricle
tissue was frozen in optimal cutting temperature compound, and transverse sections (10
μm) were generated with a cryostat and placed on glass slides. Tissue sections were
incubated with 5 μmol/l DHE at 37 °C for 20 min according to the manufacturer’s
instructions. Red fluorescence was assessed by using an Olympus FSX100 box type
fluorescence imaging device (Olympus America, Center Valley, PA). The excitation
38
wavelength was 488 nm with emission at 585 nm. Fluorescence intensity was analyzed by the use of Image J (version 1.32j) software (National Institutes of Health, USA; http://rsb.info.nih.gov/ij/).
3.3.4 Isolation of Cardiac Fibroblasts and JEG-3 Cell Experiments
Isolation of cardiac fibroblasts was carried out as previously described by Brilla and coworkers 7 with modifications as previously reported.4 Briefly, male Sprague
Dawley rats weighing 250-300 grams were used to obtain fibroblast from the hearts. The rats were anesthetized with pentobarbital (50 mg/kg), and their hearts were removed and perfused under sterile condition via the ascending aorta with Joklik's medium (Sigma-
Aldrich, St. Louis, MO) in a modified Langendorffapparatus. After 5 min of perfusion, the perfusate was placed in Joklik's medium containing 0.1% collagenase type 2
(Worthington Biochemical, Lakewood, NJ) and0.1% BSA which was circulated for 15-
25 min until the heart became flaccid. Ventricles were excised and finely cut, and shaken in Joklik's modified medium with 0.1% collagenase and 0.1%BSA for15 min. Cells
/tissue suspension was allowed to settle for 15 min and was centrifugated at 500 rpm for
10 min. The supernatant then was centrifugated at 1500 rpm for 15 min. The resulting pellet was suspended in DMEM supplemented with antibiotics
(penicillin/streptomycin/fungizone) plus 15% FBS (Hyclone, Logan, UT) and seeded onto plates and incubated for 1hr. Unattached cells were removed, and the attached fibroblasts cells were allowed to grow until confluence and then trypsinized and passaged once at 1:3 dilution. Cells were allowed to grow confluent prior to use for experimental
39
purposes. All cells used in these experiments were from passage one unless otherwise
specified. Human placental chorionic epithelial cells (JEG-3) were purchased from a
commercially available vendor (ATCC), cultured in 6-well plates, and grown to confluence in minimum essential media over 48 hours. Cells were cultured in 2.5% FBS media and rapamycin (1uM) was added into half the wells. Both control and rapamycin- treated cells were sampled after 3, 6, and 12 hours of incubation. MBG was extracted from the collected media using C18 as reported.13 Competitive immunoassays were
performed using a monoclonal anti-MBG antibody to determine the concentration of
MBG in the samples.13
3.3.5 Western Blot Analysis
Western blot analysis was performed on proteins from tissue homogenates as
previously reported.4 For the cell lysates, the cells were grown to confluence and starved
for 18 h in DMEM with 1% FBS. The cells then were treated with MBG or rapamycin for
24 h when looking for procollagen expression. The cells were washed with phosphate
buffered saline (BPS) twice and exposed to lysis buffer. For detection of Collagen-1, the
left ventricles from the heart were homogenized in ice-cold RIPA lysis buffer (pH 7.0)
Santa Cruz Biotechnology (Santa Cruz, CA; sc-24948). The homogenate was
centrifugated at 1,400g for 30 s at 4 °C. The supernatant was discarded and the pellet
fraction was resuspended in 5% sodium dodecyl sulfate (SDS) and 50 mmol/l Tris-HCl
(pH 7.4). The protein was quantified in the resuspended pellet fraction and was
solubilized in sample buffer (2% SDS, 5% β-mercaptoethanol, 20% glycerol, 0.005%
40
bromophenol blue, and 50mmol/l Tris- HCl; pH 7.0). The proteins, obtained from tissue
homogenates, were resolved on an SDS-polyacrylamide gel electrophoresis (PAGE) using Precast Ready Gels 4–15% Tris-HCl, purchased from Bio-Rad (Hercules, CA).
Ten microgram of protein per sample were loaded into each well. The proteins from the gel were electrotransferred to a nitrocellulose membrane. The membrane was blocked with 5% nonfat dry milk in 20 mmol/l Tris-HCl (pH 7.5, 150 mmol/l NaCl, and 0.1%
Tween 20). Goat anti-type 1 collagen antibody (Southern Biotech, Birmingham, AL) was used to probe for collagen-1, and secondary anti-goat antibody was purchased from
Santa Cruz Biotechnology. For detection, we used ECL and ECL plus purchased from
Amersham Biosciences. Loading conditions were controlled using anti-actin mouse monoclonal antibody (Santa Cruz Biotechnology).
3.3.6 Histology
Left ventricle sections were immediately fixed in 4% formalin buffer solution
(pH 7.2) for 18 h, dehydrated in 70% ethanol, and then embedded in paraffin and cut with a microtome. Fast green staining with sirius red (0.1%) was performed on left ventricular tissues as described previously16, and fibrosis quantified using ImageJ
software (ImageJ 1.36b, National Institutes ofHealth, USA). For confirmation of the
histological findings, quantitative determination of Collagen-1 in left ventricular homogenates was performed using Western blot (as described above).
41
3.3.7 Statistical Analysis
The results are presented as means ± s.e.m. Data were analyzed using one way
analysis of variance followed by Newman–Keuls test (intragroup analyses), by repeated
measures analysis of variance followed by Newman–Keuls test (intergroup analyses), and
by two-tailed t-test (when applicable) (GraphPad Prism software, San Diego, CA). A
two-sided P value of less than 0.05 was considered to be statistically significant.
3.4 Results
3.4.1 Effect of Rapamycin on Blood Pressure, MBG Levels
Rapamycin treatment alone demonstrated a slight elevation in systolic BP, but did
not significantly alter MBG levels compared to control animals. PNx surgery
substantially increased the heart weight/body weight ratios. PNx surgery and MBG
infusion produced sustained hypertension throughout the duration of the experiment.
PNx MBG levels were similar to the MBG levels produced by MBG infusion alone. PNx
surgery with rapamycin infusion showed a significant decrease in systolic BP by the third
week which persisted at four weeks compared to PNx alone. The PNx surgery with
rapamycin treatment also demonstrated a drastic decrease in MBG levels.
Coadministration of MBG with rapamycin did not significantly attenuate systolic BP or
plasma MBG levels. These data are summarized in Table 3.1.
42
3.4.2 Effect of Rapamycin on Cardiac Fibrosis and oxidative stress
Cardiac fibrosis was assessed in the left ventricular myocardium by histological
analysis (sirius red with fast green staining) and collagen 1 expression determined by
Western blot. Both PNx and MBG infusion resulted in substantial increases of collagen
expression and cardiac scarring, while PNx with rapamycin infusion drastically lowered
these effects (Figure 3-1 A and B). Coadministration of rapamycin with MBG did not alter MBG induced cardiac fibrosis (Figure 3-1 A, and B). The increase in cardiac fibrosis in PNx and MBG treated animals was accompanied with an increase in cardiac oxidative stress as measured by DHE staining (Figure 3-2). Treatment with rapamycin did not alter oxidative stress in these animals.
3.4.3 Effect of Rapamycin on Fibroblast Procollagen Expression
Cultured cardiac fibroblasts treated with 1 and 100 nM of MBG resulted in a significant increase in procollagen 1 expression determined by Western blot (Figure 3.3).
Treatment with rapamycin (10.9 and 109 pM) significantly attenuated MBG (1 and 100 nM) induced procollagen 1 expression (Figure 3-3.) The concentrations of rapamycin chosen have been reported to have little effect on cell viability 17.
43
3.4.4 Effect of Rapamycin on MBG Production by JEG-3 Cells
Cultured human placental chorionic epithelial cells (JEG-3), which produce
MBG, were treated with 1 µM of rapamycin to test the effects on MBG production.
Rapamycin treatment (1 uM) at 3 and 6 hours significantly reduced MBG production compared to controls (84 vs 60 pmol/g protein, p<0.01; and 243 vs 116, pmol/g protein, p<0.01, Figure 3-4).
3.5 Discussion
The mTOR pathway has been shown to play a pivotal role in several different forms of renal disease.9 Treatment with rapamycin attenuates many of the physiological
changes associated with a decrease in renal function, including interstitial fibrosis.9 Our
current work demonstrates that rapamycin treatment in the setting of experimental uremic
cardiomyopathy significantly reduces cardiac fibrosis. This is in support of a recent
report with similar findings in a murine model of uremic cardiomyopathy.10 We have
also shown that fibroblast treatment with rapamycin drastically reduced procollagen
production in the presence of MBG. Importantly, rapamycin also drastically lowered
MBG levels in both the PNx model and in human placental chorionic epithelial cells
(JEG-3).
Recent work in animal models of renal disease has provided compelling evidence
for the involvement of mTORC1 in the generation of fibrosis. In an animal model of
unilateral obstructive nephropathy, as well as in fibroblasts, the profibrotic cytokine
44
TGF-β was shown to activate mTORC1 acting through a PI3K pathway. 18 In human
fibroblasts, the mTOR pathway has been shown to regulate collagen type I production. 19
Treatment with rapamycin has been shown to decrease TGF-β, fibroblast proliferation, and renal fibrosis. 18, 20, 21 In support of our results, rapamycin treatment significantly
decreased cardiac fibrosis as evaluated by trichrome staining in a murine model of uremic
cadiomyopathy.10 Similar results were reported using a transverse aortic constriction
model. 22
We have shown that MBG causes many of the pathophysiological changes
associated with experimental uremic cardiomyopathy including cardiac fibrosis 5, and
that MBG induces cardiac fibroblasts to produce collagen.4 The transcription factor
Friend leukemia integration-1 (Fli-1) acts as a negative regulator of collagen production
23, 24, and activation of protein kinase C-delta (PKC- δ) can phosphorylate Fli-1 to promote collagen synthesis. 25 We have recently reported that MBG induces
translocation of PKC-delta, which phosphorylates Fli-1 and leads to an increase in
collagen synthesis.26 Signaling through PKC-delta has been shown to activate the mTOR pathway.27
Treatment with rapamycin significantly reduced circulating MBG levels
compared to PNx animals. In addition, treatment with rapamycin in JEG-3 cells, which
produce MBG, resulted in a 52% reduction in MBG levels after six hours of treatment.
Endogenous cardiotonic steroids have been postulated to be synthesized from the classic
steroidogenesis pathway through cholesterol side-chain cleavage and pregnenolone precursors.2 Though this theory is still upheld for other cardiotonic steroids such as
45
ouabain, there have been controversial results with regard to MBG production.2 In toads, the biosynthesis of MBG occurs via the bile acid pathway form cholanic acids.2
Rapamycin acts as a competitive inhibitor of CYP27A1, a key rate-limiting enzyme of the bile acid pathway. Our data provides preliminary evidence indicating that the drastic reduction in MBG levels in both PNx animals and JEG-3 cells may be due to competitive inhibition of CYP27A1. Thus, in the setting of experimental uremic cardiomyopathy, rapamycin may have a duel effect of both inhibiting cardiac fibrosis and reducing MBG production.
Importantly, we did not see a significant reduction in cardiac fibrosis or MBG levels with combined MBG infusion and rapamycin treatment. We also did not see a reduction in oxidant stress in PNx animals treated with rapamycin or combined MBG and rapamycin treatment. We speculate that rapamycin was unable to provide a therapeutic effect in the setting of continuous MBG infusion. Furthermore, MBG induced ROS production may precede mTOR activation. Future experiments are warranted in order to determine if MBG is produced by the bile acid pathway in mammals, and if higher doses of rapamycin would overcome the effects of continuous MBG infusion.
In conclusion, the mTOR pathway has been implicated in the generation of renal fibrosis during renal failure. Our results suggest that the mTOR pathway may have a significant impact in the generation of cardiac fibrosis. Treatment with rapamycin may provide a novel therapy for reducing MBG levels and cardiac fibrosis in the setting of uremic cardiomyopathy.
46
3.6 Manuscript References 1. Sarnak MJ, Levey AS, Schoolwerth AC, Coresh J, Culleton B, Hamm LL,
McCullough PA, Kasiske BL, Kelepouris E, Klag MJ, Parfrey P, Pfeffer M, Raij
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3. Kolmakova EV, Haller ST, Kennedy DJ, Isachkina AN, Budny GV, Frolova EV,
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Malhotra D, Fedorova OV, Kashkin VA, Bagrov AY, Shapiro JI.
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5. Kennedy DJ, Vetteth S, Periyasamy SM, Kanj M, Fedorova L, Khouri S, Kahaleh
MB, Xie Z, Malhotra D, Kolodkin NI, Lakatta EG, Fedorova OV, Bagrov AY,
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6. Haller ST, Kennedy DJ, Shidyak A, Budny GV, Malhotra D, Fedorova OV,
Shapiro JI, Bagrov AY. Monoclonal Antibody Against Marinobufagenin Reverses
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8. Sarbassov DD, Ali SM, Kim DH, Guertin DA, Latek RR, Erdjument-Bromage H,
Tempst P, Sabatini DM. Rictor, a novel binding partner of mTOR, defines a
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cytoskeleton. Curr Biol. 2004;14(14):1296-1302.
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10. Siedlecki AM, Jin X, Muslin AJ. Uremic cardiac hypertrophy is reversed by
rapamycin but not by lowering of blood pressure. Kidney Int. 2009;75(8):800-
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11. Gueguen Y, Ferrari L, Souidi M, Batt AM, Lutton C, Siest G, Visvikis S.
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12. Inman SR, Davis NA, Olson KM, Lukaszek VA, McKinley MR, Seminerio JL.
Rapamycin preserves renal function compared with cyclosporine A after
ischemia/reperfusion injury. Urology. 2003;62(4):750-754.
13. Fedorova OV, Simbirtsev AS, Kolodkin NI, Kotov AY, Agalakova NI, Kashkin
VA, Tapilskaya NI, Bzhelyansky A, Reznik VA, Frolova EV, Nikitina ER, Budny
GV, Longo DL, Lakatta EG, Bagrov AY. Monoclonal antibody to an endogenous
bufadienolide, marinobufagenin, reverses preeclampsia-induced Na/K-ATPase
inhibition and lowers blood pressure in NaCl-sensitive hypertension. J Hypertens.
2008;26(12):2414-2425.
14. Zanetti M, d'Uscio LV, Peterson TE, Katusic ZS, O'Brien T. Analysis of
superoxide anion production in tissue. Methods Mol Med. 2005;108:65-72.
15. Takimoto E, Champion HC, Li M, Ren S, Rodriguez ER, Tavazzi B, Lazzarino G,
Paolocci N, Gabrielson KL, Wang Y, Kass DA. Oxidant stress from nitric oxide
synthase-3 uncoupling stimulates cardiac pathologic remodeling from chronic
pressure load. J Clin Invest. 2005;115(5):1221-1231.
16. Lopez-De Leon A, Rojkind M. A simple micromethod for collagen and total
protein determination in formalin-fixed paraffin-embedded sections. J Histochem
Cytochem. 1985;33(8):737-743.
17. Poulalhon N, Farge D, Roos N, Tacheau C, Neuzillet C, Michel L, Mauviel A,
Verrecchia F. Modulation of collagen and MMP-1 gene expression in fibroblasts
by the immunosuppressive drug rapamycin. A direct role as an antifibrotic agent?
J Biol Chem. 2006;281(44):33045-33052.
49
18. Wang S, Wilkes MC, Leof EB, Hirschberg R. Noncanonical TGF-beta pathways,
mTORC1 and Abl, in renal interstitial fibrogenesis. Am J Physiol Renal
Physiol.298(1):F142-149.
19. Shegogue D, Trojanowska M. Mammalian target of rapamycin positively
regulates collagen type I production via a phosphatidylinositol 3-kinase-
independent pathway. J Biol Chem. 2004;279(22):23166-23175.
20. Shillingford JM, Piontek KB, Germino GG, Weimbs T. Rapamycin ameliorates
PKD resulting from conditional inactivation of Pkd1. J Am Soc
Nephrol.21(3):489-497.
21. Lloberas N, Cruzado JM, Franquesa M, Herrero-Fresneda I, Torras J, Alperovich
G, Rama I, Vidal A, Grinyo JM. Mammalian target of rapamycin pathway
blockade slows progression of diabetic kidney disease in rats. J Am Soc Nephrol.
2006;17(5):1395-1404.
22. Gao XM, Wong G, Wang B, Kiriazis H, Moore XL, Su YD, Dart A, Du XJ.
Inhibition of mTOR reduces chronic pressure-overload cardiac hypertrophy and
fibrosis. J Hypertens. 2006;24(8):1663-1670.
23. Czuwara-Ladykowska J, Shirasaki F, Jackers P, Watson DK, Trojanowska M. Fli-
1 inhibits collagen type I production in dermal fibroblasts via an Sp1-dependent
pathway. J Biol Chem. 2001;276(24):20839-20848.
24. Wang Y, Fan PS, Kahaleh B. Association between enhanced type I collagen
expression and epigenetic repression of the FLI1 gene in scleroderma fibroblasts.
Arthritis Rheum. 2006;54(7):2271-2279.
50
25. Jinnin M, Ihn H, Yamane K, Mimura Y, Asano Y, Tamaki K. Alpha2(I) collagen
gene regulation by protein kinase C signaling in human dermal fibroblasts.
Nucleic Acids Res. 2005;33(4):1337-1351.
26. Elkareh J, Periyasamy SM, Shidyak A, Vetteth S, Schroeder J, Raju V, Hariri IM,
El-Okdi N, Gupta S, Fedorova L, Liu J, Fedorova OV, Kahaleh MB, Xie Z,
Malhotra D, Watson DK, Bagrov AY, Shapiro JI. Marinobufagenin induces
increases in procollagen expression in a process involving protein kinase C and
Fli-1: implications for uremic cardiomyopathy. Am J Physiol Renal Physiol.
2009;296(5):F1219-1226.
27. Minhajuddin M, Bijli KM, Fazal F, Sassano A, Nakayama KI, Hay N, Platanias
LC, Rahman A. Protein kinase C-delta and phosphatidylinositol 3-kinase/Akt
activate mammalian target of rapamycin to modulate NF-kappaB activation and
intercellular adhesion molecule-1 (ICAM-1) expression in endothelial cells. J Biol
Chem. 2009;284(7):4052-4061.
51
3.7 Table and Figure Legends
Table 3.1. Effects of rapamycin on physiological measurements after PNx or infusion of MBG.
Sham refers to animals subject to sham surgery; PNx refers to partial nephrectomy; PNx
+ Rapa refers to PNx surgery and rapamycin infusion using minipumps; Rapa refers to rapamycin infusion using minipumps; MBG + Rapa refers to coadministration of MBG and rapamycin using minipumps; and MBG refers to MBG infusion using minipumps.*p<0.05 vs sham, †p<0.01 vs sham, §p<0.01 vs PNx, ‡p<0.01 vs MBG,
║p<0.01 vs PNx + Rapa, ¶p<0.01 vs MBG + Rapa
Figure 3-1. A, Representative (top) and quantitative analysis of collagen 1 (mean±SEM)
Western blots performed on cardiac tissue from the different groups. Actin was used as a loading control. B, Representative Sirius red and Fast green stained photomicrographs obtained from cardiac tissue derived from the different experimental groups. Amount of fibrosis expressed as mean±SEM measured using computer-assisted morphometry, as we have previously described 5. Sham refers to animals subject to sham surgery (n=8); PNx refers to partial nephrectomy (n=10); PNx + Rapa refers to PNx surgery and rapamycin infusion using minipumps (n=6); Rapa refers to rapamycin infusion using minipumps
(n=8); MBG + Rapa refers to coadministration of MBG and rapamycin using minipumps
(n=8); and MBG refers to MBG infusion using minipumps (n=8). *p<0.05 vs Sham,
**p<0.01 vs Sham, #p<0.01 vs PNx, ##p<0.01 vs Rapa, †p<0.05 vs Rapa.
52
Figure 3-2. Effects of rapamycin treatment on cardiac levels of oxidative stress assessed by DHE fluorescence intensity: (upper panels, representative measurements; lower panel
– quantitative measurements, mean±SEM of 4 densitometry determinations). Sham refers to animals subject to sham surgery; PNx refers to partial nephrectomy; PNx + Rapa refers to PNx surgery and rapamycin infusion using minipumps; Rapa refers to rapamycin infusion using minipumps; MBG + Rapa refers to coadministration of MBG and rapamycin using minipumps; and MBG refers to MBG infusion using minipumps.
*p<0.01 vs Sham, **p<0.01 vs Rapa.
Figure 3-3. Representative Western blot against procollagen 1 derived from cardiac fibroblasts treated with MBG (1 or 100 nM), rapamycin (10.9 or 109 pM), or a combination with the corresponding quantitative data shown as the mean±SEM of 5 experiments. *P<0.01 vs control, **p<0.01 vs MBG 100 nM, #P<0.01 vs MBG 1 nM
Figure 3-4. MBG production in JEG-3 cells after incubation with 1uM rapamycin for 3 and 6 hours. *p<0.05 vs control, **p<0.01 vs control.
53
3.8 Table and Figures
Table 3.1
54
Figure 3-1.
B Collagen-1
A Sham Rapa PNx Actin
PNx + Rapa MBG MBG + Rapa
55
Figure 3-2.
Sham Rapa PNx
PNx-Rapa MBG MBG-Rapa
56
Figure 3-3.
Procollagen-1
Actin
57
Figure 3-4.
58
Chapter 4 – Manuscript
Title:
Platelet Activation in Patients with Atherosclerotic Renal Artery Stenosis Undergoing Stent Revascularization
Authors:
Steven T. Haller1, MS, Satjit Adlakha1, D.O., Grant Reed2, M.D., Pamela Brewster1, MA, David Kennedy3, PhD, Mark W. Burket1, M.D., FACC, William Colyer1, M.D., Haifeng Yu1, MS, Dong Zhang1, MS, Joseph I. Shapiro1, MD, Christopher J. Cooper1, MD, FACC
1Department of Medicine, University of Toledo Collage of Medicine, Toledo, OH
2Brigham and Women’s Hospital, Boston, MA
3Cleveland Clinic Foundation, Cleveland, OH
Corresponding Author:
Steven T. Haller, University of Toledo, Collage of Medicine, 3000 Arlington Ave., Toledo, OH
Published in the Clinical Journal of the American Society of Nephrology, 2011 Sep;6(9):2185-91. Epub 2011 Aug 4.
59
4.1 Abstract
Background and Objectives: Soluble CD40 ligand (sCD40L) is a marker of platelet activation; whether platelet activation occurs in the setting of renal artery stenosis and stenting is unknown. Additionally, the effect of embolic protection devices and glycoprotein IIb/IIIa inhibitors on platelet activation during renal artery intervention is unknown.
Design, setting, participants, & measurements: Plasma levels of sCD40L were
measured in healthy controls, patients with atherosclerosis without renal stenosis, and
patients with renal artery stenosis before, immediately after, and 24hr after renal artery
stenting.
Results: Soluble CD40L levels were higher in renal artery stenosis patients than normal
controls (347.5 ± 27.0 vs 65.2 ± 1.4 pg/ml, p<0.001), but were similar to patients with
atherosclerosis without renal artery stenosis. Platelet rich emboli were captured in 26%
(9/35) of embolic protection device patients and in these patients sCD40L was elevated
prior to the procedure. Embolic protection device use was associated with a non-
significant increase in sCD40L whereas sCD40L declined with abciximab post-procedure
(324.9 ± 42.5 vs 188.7 ± 31.0 pg/ml, p=0.003) and at 24hrs.
Conclusions: Atherosclerotic renal artery stenosis is associated with platelet activation
but this appears to be related to atherosclerosis, not renal artery stenosis specifically.
Embolization of platelet rich thrombi is common in renal artery stenting and is inhibited
with abciximab.
60
4.2 Introduction
Platelet activation leading to thrombus formation is a well-described complication
of coronary artery disease, yet its occurrence in renal artery stenosis (RAS) is unknown. 1-
4 RAS is a major cause of secondary hypertension and an important cause of renal
failure.1-3, 5, 6 Although the utility of stent revascularization in patients with RAS is
uncertain, several studies suggest that at least a portion of patients develop a loss of
kidney function post-procedure. 1-3 6, 7
Soluble CD40 ligand (sCD40L) is expressed and secreted by platelets after
activation and plays a vital role in the immune, inflammatory, and coagulative responses
following injury or stress and in the setting of transplantation has been linked to renal
fibrosis .8-15 Moreover, high levels of sCD40L correlate with cardiovascular events in patients with unstable coronary syndromes. 13, 16-18 GP IIb/IIIa inhibitors may lower the
level of platelet activation in vitro and the level of sCD40L released from platelets upon
activation.19, 20 A recent report from our group has demonstrated that the use of a
GPIIb/IIIa inhibitor in combination with an embolic protection device (EPD) during renal
artery stenting may improve renal function following the revascularization procedure.21
However the relationship between platelet activation and patient outcome following renal artery stenting is uncertain.
On this background, the goals of the present study were to determine 1) if platelet activation is associated with atherosclerotic RAS, and 2) whether platelet activation occurs during renal artery stenting, and 3) if platelet thrombus formation captured by the
EPD correlates with systemic platelet activation .
61
4.3 Materials and Methods
The study, ClinicalTrials.gov identifier NCT00234585, was conducted with
funding provided by the sponsors, but study conduct, analysis, and reporting performed
independent of the sponsors. ICH good clinical practice guidelines were followed with
patients providing informed consent in an IRB approved protocol.
Platelet activation levels from the RAS patients were compared to 30 healthy
controls and, 30 patients with atherosclerosis undergoing coronary angiography, but free
of RAS. A total of 100 RAS patients were recruited from 7 sites. Inclusion required a
history of hypertension, renal insufficiency, heart failure, or angina with poorly
controlled hypertension and also the presence of 1 or more stenoses, ≥ 50% and <100, treatable with the EPD. RAS patients were randomized to the following allocations: 1/2 to Angioguard, 1/2 to no Angioguard; 1/2 to abciximab, 1/2 to placebo infusion yielding four groups: control, Angioguard only, abciximab only, and Angioguard with abciximab.
4.3.1 Pre-procedural care
In patients with RAS prior to double-blinded administration of Abciximab or placebo, systolic blood pressure was lowered to <160 mmHg. The target ACT was 275
seconds, if randomized to the EPD device an ACT of >300 seconds was required. A
bolus of 0.25 mg/kg abciximab (or placebo) was administered 5 minutes before crossing
the lesion, and was followed by an infusion at 0.125 µg/kg/min (maximum 10 µg /min) for 12 hours.
62
4.3.2 Central Laboratory Analysis
The blinded analysis of EPD contents was performed by the CV Path core lab
(Gaithersburg, MD). Platelet emboli consisted of layered platelet aggregates with
varying amounts of entrapped leukocytes and fibrin as evidence on hematoxylin and
eosin stained sections.22 Glomerular filtration rate (GFR), calculated from the modified
MDRD equation23, was used as the primary measure of renal function. Creatinine was
measured by a modified Jaffe reaction using the IDMS-traceable assay at the University
of Minnesota Core Lab for all subjects.
4.3.3 Blood Collection
Peripheral venous blood was collected at baseline, immediate post, and 24hr post procedure in lithium heparin plasma separator tubes, spun at 1000 x g for 15 minutes, and frozen at –80◦C until batch analysis.
4.3.4 Measurement of soluble CD40 ligand
Plasma levels of soluble CD40 ligand (sCD40L) were measured by enzyme linked immunosorbent assay (ELISA, R&D Systems; Minneapolis, MN). The ELISA kit
had intra-assay and interassay coefficients of 5% and 6%, respectively. The average
minimum detectible amount of sCD40L was 4.2 pg/ml.
63
4.3.5 Statistical Analysis
Study data are presented as continuous (mean±SEM) and categorical data.
Statistical analysis was performed on subjects with complete data for platelet activation measurements at the baseline, immediate post, and 24hr post procedure time points
(n=84). SAS one-way ANOVA were used to test for significance among groups.
Paired t-tests and Fisher’s PLSD post hoc tests were used to test for significance between groups. Unpaired t-tests were used to test for significance between the normal subjects, patient controls, and the RAS patients. Significance was defined as P<0.05. All analyses were performed in SAS or JMP.
4.4 Results
Baseline characteristics of the normal controls (n=30), atherosclerotic controls
(n=30), and the RAS patients (n=84), are shown in Table 4.1. The RAS patients had a significantly higher level of sCD40L compared to normal controls (347.5 ± 27.1 vs 65.2
± 1.4 pg/ml, P<0.001) (Figure 4-1). However, sCD40L levels were similar when compared to the patients with atherosclerosis who were free of renal artery stenosis
(347.5 ± 27.1 vs 335.2 ± 38.6 pg/ml, P=0.79) (Figure 4-1). Soluble CD40L, either at baseline or after the stenting, was not associated with baseline GFR or subsequent changes in kidney function.
64
4.4.1 EPD Content, Platelet Embolization, and sCD40L
Twenty six percent (9/35) of the patients that received the Angioguard had platelet rich emboli captured within the filter. In these patients with platelet rich emboli, sCD40L levels were higher than in patients without platelet emboli both before (497.9 ±
105.0 vs 313.7 ± 28.4 pg/ml, p=0.02) and after the procedure (443.3 ± 111.3 vs 232.2 ±
32.4 pg/ml, p=0.02) (Figure4-2)
4.4.2 Effect of Distal Protection and Drug Treatment
Patients with RAS randomized to abciximab had a significant decrease in
sCD40L levels immediately following the procedure (324.9 ± 42.5 vs 188.7 ± 31.0 pg/ml,
p=0.003), which persisted at 24hrs (324.9 ± 42.5 vs 181.2 ± 19.3 pg/ml, p=0.002) (Figure
4.3). In patients randomized to the Angioguard, sCD40L levels rose slightly immediately
following the procedure and at 24hrs (p=0.90) (Figure 4-3). Patients randomized to both
the Angioguard device and abciximab showed a significant decrease in sCD40L
immediately following the procedure (322.8 ± 35.2 vs 203.6 ± 33.1 pg/ml, p=0.03) but
this difference was no longer significant at 24 hours (Figure 4-3).
4.4.3 Effect of Abciximab and Clopidogrel
Clopiogrel use was not associated with lower sCD40L at baseline. For patients on
clopidogrel before intervention, sCD40L levels rose slightly immediately following the
procedure and decreased at 24hrs (p=0.53). For patients randomized to Abciximab and
not taking clopidogrel, sCD40L levels decreased significantly immediately following the
procedure (310.5 ± 33.0 vs 195.2 ± 31.3 pg/ml, p=0.008), which persisted at 24hrs (310.5 65
± 33.0 vs 173.1 ± 18.7 pg/ml, p<0.001. Patients on clopidogrel and randomized to
Abciximab showed a significant decrease in sCD40L at immediate post procedure (346.3
± 48.1 vs 198.3 ± 30.6, p=0.02). Similar effects were seen in patients that were
prescribed clopidogrel on the day of procedure.
4.5 Discussion
Platelet activation is a major cause of events and complications in coronary artery disease and with coronary revascularization. 24, 25 The use of platelet inhibitors during
coronary stenting reduces the potentially harmful effects of platelet activation including
abrupt vessel occlusion, myocardial infarction, and stent thrombosis. 25 To date, the
extent of platelet activation, and effect of anti-platelet therapies in the setting of renal
artery stenting has not been established. Thus, in the current study we sought to
determine whether atherosclerotic renal artery stenosis was associated with platelet
activation and the effect(s) of embolic protection and or use of platelet inhibitors on
markers of platelet activation.
Increased platelet activation is associated with a variety of vascular disorders
including acute coronary syndromes, stable coronary artery disease, and restenosis
following percutaneous coronary intervention. 26, 27 Soluble CD40L is a particularly
attractive marker for platelet activation since it is shed from the surface of activated
platelets, is easily measured, and meaningfully participates in a number of important
biologic processes including activation of immunity and thrombosis.28 The current study
found increased levels of sCD40L in the setting of RAS, however, this appears to be a
non-specific association with atherosclerosis in general as opposed to being attributable 66
to RAS specifically. More importantly though increased levels of sCD40L prior to the
procedure were more likely to have embolization of platelet-rich thrombi and these patients had persistently elevated levels of sCD40L after the procedure. This finding may represent a potentially modifiable feature denoting increased risk for patients referred for renal artery revascularization.
The current study also demonstrated that abciximab effectively inhibits platelet activation, as denoted by substantial suppression of sCD40L, up to 24 hrs following the procedure. Others have also observed the ability of GPIIb/IIIa inhibitors to lower levels of sCD40L in settings such as acute coronary syndromes and in STEMI patients undergoing coronary intervention. 29, 30 The current finding extends the prior observation that a GPIIb/IIIa inhibitor, when combined with an embolic protection device to capture atheroembolic debri, resulted in the most favorable renal function outcome. 21
The suppression of sCD40L release, observed with abciximab administration in
the current study, creates a plausible biologic pathway to explain the observation that
abciximab use was associated with improved renal function after stenting. In the kidney
the sCD40L/CD40 may be directly responsible for renal injury. Previously, others have
shown that angiotensin II stimulates release of renal TGF-β that in turn increases expression of the CD40 receptor on the proximal tubule of the kidney. 31 Pontrelli et al.
has shown that CD40 cross-linking on proximal tubular epithelial cells is pro-
inflammatory and induces fibrosis by stimulating the expression of plasminogen activator
inhibitor-1 (PAI-1) acting through a signaling pathway which is independent of the
proinflammatory signaling effects of CD40L.15 In addition, activation of the CD40
67
receptor results in infiltration of inflammatory cells into the interstitium of the kidney
through monocyte chemoattractant protein-1 (MCP-1) and intercellular adhesion molecule-1 (ICAM-1) expression.32 IL-8 amplifies CD40/CD154-mediated ICAM-1 production via the CXCR-1 receptor and p38-MAPK pathway in human renal proximal tubule cells.32 Furthermore, inhibition of the CD40/CD40L significantly decreased the
severity of renal injury in an animal model of chronic proteinuric renal disease.33 Thus, it
is conceivable that in patients with renal ischemia 1) the CD40 receptor is over-expressed
due to angiotensin II stimulation, 2) sCD40L shed by locally activated platelets may
activate the receptor and stimulate peritubular fibrosis in a manner independent of renal
blood flow or ischemia, and 3) this process may be accelerated at the time of a stent
procedure. In this regard the association between the GP IIb/IIIa inhibitor Abciximab
and improved renal function outcomes observed in the RESIST study21 may be
attributable to the drug’s effects in suppressing sCD40L as opposed to an effect on
thrombosis per se.
An observation from the RESIST study was that the EPD, when used without
abciximab, did not appear to improve renal function despite capturing debris. In the
current study we saw a slight increase in platelet activation with the use of the EPD
occurring immediately after the procedure, although this increase was not statistically
significant. Conceivably the EPD may slow blood flow in the vessel, provide a surface
upon which platelets can aggregate, and increase local platelet activation an effect
inhibited by the GPIIb/IIIa inhibitor. Admittedly the observed increase in circulating
levels of sCD40L with the use of the EPD was not statistically significant, however, it
68
may be unrealistic to expect that effects occurring on the surface of an EPD would be
detected systemically.
Several studies suggest a benefit of reducing platelet activation with loading doses
of 300-600 mg of clopidogrel prior to coronary interventions.34-36 However in the current
study pre-treatment with clopidogrel or clopidogrel administration on the day of
procedure did not significantly effect sCD40L levels. This may result from confounding
since patients were not randomized to clopidogrel treatment and had a significantly
higher prevalence of coronary artery and peripheral vascular disease, which may account
for the lack of difference observed in sCD40L levels. Work by Azar et al. reported a
reduction in sCD40L at a clopidogrel dose of 75 mg/day when preceded by a loading
dose of 300 mg in patients with stable CAD.37 Others though have failed to demonstrate
an effect of clopidogrel on levels of sCD40L.38
Increased levels of circulating sCD40L and the impact on renal function in the
setting of RAS remain speculative. Future clinical trials should address the effect of
sCD40L inhibition on distal embolization and renal function with long term follow-up.
The current study provides a foundation for exploring the role of CD40/CD40L signaling
and the generation of renal fibrosis during ischemic renal injury.
The following limitations of our study warrant mentioning: The current study
utilized sCD40L as the key measure of platelet activation. We did not measure sCD40L
at one month nor do we have longer-term follow-up of renal function beyond one month.
Thus, it remains uncertain whether other indices of platelet activation would provide
69
additional insights, or whether longer term follow up would have yielded similar results
for kidney function.
Atherosclerotic RAS is associated with increased platelet activation, but this increase appears to be attributable to atherosclerosis in general, not RAS specifically.
However, in patients with higher levels of platelet activation prior to the procedure, embolization of platelet rich thrombi is more common. Abciximab effectively inhibits platelet activation and sCD40L release, a mechanism that may explain the beneficial effect on renal function one month after the procedure that has been previously observed.
70
4.6 Manuscript References
1. Levin A, Linas S, Luft FC, Chapman AB, Textor S: Controversies in renal artery
stenosis: a review by the American Society of Nephrology Advisory Group on
Hypertension. Am J Nephro 27: 212-220. 2007
2. Balk E, Raman G, Chung M, Ip S, Tatsioni A, Alonso A, Chew P, Gilbert SJ, Lau
J: Effectiveness of management strategies for renal artery stenosis: a systematic
review. Ann Intern Med 145: 901-912. 2006
3. Olin JW: Survival in atherosclerotic renal artery stenosis: its all about renal
function, or is it? Catheter Cardiovasc Interv 69: 1048-1049. 2007
4. Arthurs Z, Starnes B, Cuadrado D, Sohn V, Cushner H, Andersen C: Renal artery
stenting slows the rate of renal function decline. J Vasc Surg 45: 726-731. 2007
5. Safian RD, Textor SC: Renal-artery stenosis. N Engl J Med 344: 431-442. 2001
6. Hirsch AT, Haskal ZJ, Hertzer NR, Bakal CW, Creager MA, Halperin JL,
Hiratzka LF, Murphy WR, Olin JW, Puschett JB, Rosenfield KA, Sacks D,
Stanley JC, Taylor LM, Jr., White CJ, White J, White RA, Antman EM, Smith
SC, Jr., Adams CD, Anderson JL, Faxon DP, Fuster V, Gibbons RJ, Hunt SA,
Jacobs AK, Nishimura R, Ornato JP, Page RL, Riegel B: ACC/AHA 2005
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Surgery, Society for Cardiovascular Angiography and Interventions, Society for
Vascular Medicine and Biology, Society of Interventional Radiology, and the
ACC/AHA Task Force on Practice Guidelines (Writing Committee to Develop
Guidelines for the Management of Patients With Peripheral Arterial Disease):
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Rehabilitation; National Heart, Lung, and Blood Institute; Society for Vascular
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Foundation. Circulation 113: e463-654. 2006
7. Cooper CJ, Murphy TP, Matsumoto A, Steffes M, Cohen DJ, Jaff M, Kuntz R,
Jamerson K, Reid D, Rosenfield K, Rundback J, D'Agostino R, Henrich W,
Dworkin L: Stent revascularization for the prevention of cardiovascular and renal
events among patients with renal artery stenosis and systolic hypertension:
rationale and design of the CORAL trial. Am Heart J 152: 59-66. 2006
8. Inwald DP, McDowall A, Peters MJ, Callard RE, Klein NJ: CD40 is
constitutively expressed on platelets and provides a novel mechanism for platelet
activation. Circ Res 92: 1041-1048. 2003
9. Chakrabarti S, Varghese S, Vitseva O, Tanriverdi K, Freedman JE: CD40 ligand
influences platelet release of reactive oxygen intermediates. Arterioscler Thromb
Vasc Biol 25: 2428-2434. 2005
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10. Henn V, Slupsky JR, Grafe M, Anagnostopoulos I, Forster R, Muller-Berghaus G,
Kroczek RA: CD40 ligand on activated platelets triggers an inflammatory
reaction of endothelial cells. Nature 391: 591-594. 1998
11. Freedman JE: CD40-CD40L and platelet function: beyond hemostasis. Circ Res
92: 944-946. 2003
12. Andre P, Prasad KS, Denis CV, He M, Papalia JM, Hynes RO, Phillips DR,
Wagner DD: CD40L stabilizes arterial thrombi by a beta3 integrin--dependent
mechanism. Nat Med 8: 247-252. 2002
13. Mason PJ, Chakrabarti S, Albers AA, Rex S, Vitseva O, Varghese S, Freedman
JE: Plasma, serum, and platelet expression of CD40 ligand in adults with
cardiovascular disease. Am J Cardiol 96: 1365-1369. 2005
14. Santilli F, Davi G, Consoli A, Cipollone F, Mezzetti A, Falco A, Taraborelli T,
Devangelio E, Ciabattoni G, Basili S, Patrono C: Thromboxane-dependent CD40
ligand release in type 2 diabetes mellitus. J Am Coll Cardiol 47: 391-397. 2006
15. Pontrelli P, Ursi M, Ranieri E, Capobianco C, Schena FP, Gesualdo L,
Grandaliano G: CD40L proinflammatory and profibrotic effects on proximal
tubular epithelial cells: role of NF-kappaB and lyn. J Am Soc Nephrol 17: 627-
636. 2006
16. Nannizzi-Alaimo L, Rubenstein MH, Alves VL, Leong GY, Phillips DR, Gold
HK: Cardiopulmonary bypass induces release of soluble CD40 ligand.
Circulation 105: 2849-2854. 2002 73
17. Kritharides L, Lau GT, Freedman B: Soluble CD40 ligand in acute coronary
syndromes. N Engl J Med 348: 2575-2577. 2003
18. Burdon KP, Langefeld CD, Beck SR, Wagenknecht LE, Carr JJ, Rich SS,
Freedman BI, Herrington D, Bowden DW: Variants of the CD40 gene but not of
the CD40L gene are associated with coronary artery calcification in the Diabetes
Heart Study (DHS). Am Heart J 151: 706-711. 2006
19. Welt FG, Rogers SD, Zhang X, Ehlers R, Chen Z, Nannizzi-Alaimo L, Phillips
DR, Simon DI: GP IIb/IIIa inhibition with eptifibatide lowers levels of soluble
CD40L and RANTES after percutaneous coronary intervention. Catheter
Cardiovasc Interv 61: 185-189. 2004
20. Nannizzi-Alaimo L, Alves VL, Phillips DR: Inhibitory effects of glycoprotein
IIb/IIIa antagonists and aspirin on the release of soluble CD40 ligand during
platelet stimulation. Circulation 107: 1123-1128. 2003
21. Cooper CJ, Haller ST, Colyer W, Steffes M, Burket MW, Thomas WJ, Safian R,
Reddy B, Brewster P, Ankenbrandt MA, Virmani R, Dippel E, Rocha-Singh K,
Murphy TP, Kennedy DJ, Shapiro JI, D'Agostino RD, Pencina MJ, Khuder S:
Embolic protection and platelet inhibition during renal artery stenting. Circulation
117: 2752-2760. 2008
22. Burches B, Karnicki K, Wysokinski W, McBane RD, 2nd: Immunohistochemistry
of thrombi following iliac venous stenting: a novel model of venous thrombosis.
Thromb Haemost 96: 618-622. 2006
74
23. Stevens LA, Coresh J, Greene T, Levey AS: Assessing kidney function--
measured and estimated glomerular filtration rate. N Engl J Med 354: 2473-2483.
2006
24. Anand SX, Kim MC, Kamran M, Sharma SK, Kini AS, Fareed J, Hoppensteadt
DA, Carbon F, Cavusoglu E, Varon D, Viles-Gonzalez JF, Badimon JJ, Marmur
JD: Comparison of platelet function and morphology in patients undergoing
percutaneous coronary intervention receiving bivalirudin versus unfractionated
heparin versus clopidogrel pretreatment and bivalirudin. Am J Cardiol 100: 417-
424. 2007
25. Garg R, Uretsky BF, Lev EI: Anti-platelet and anti-thrombotic approaches in
patients undergoing percutaneous coronary intervention. Catheter Cardiovasc
Interv 70: 388-406. 2007
26. Santilli F, Basili S, Ferroni P, Davi G: CD40/CD40L system and vascular disease.
Intern Emerg Med 2: 256-268. 2007
27. Turker S, Guneri S, Akdeniz B, Ozcan MA, Baris N, Badak O, Kirimli O, Yuksel
F: Usefulness of preprocedural soluble CD40 ligand for predicting restenosis after
percutaneous coronary intervention in patients with stable coronary artery disease.
Am J Cardiol 97: 198-202. 2006
28. Antoniades C, Bakogiannis C, Tousoulis D, Antonopoulos AS, Stefanadis C: The
CD40/CD40 ligand system: linking inflammation with atherothrombosis. J Am
Coll Cardiol 54: 669-677. 2009
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29. Furman MI, Krueger LA, Linden MD, Fox ML, Ball SP, Barnard MR, Frelinger
AL, 3rd, Michelson AD: GPIIb-IIIa antagonists reduce thromboinflammatory
processes in patients with acute coronary syndromes undergoing percutaneous
coronary intervention. J Thromb Haemost 3: 312-320. 2005
30. Dominguez-Rodriguez A, Abreu-Gonzalez P, Avanzas P, Bosa-Ojeda F, Samimi-
Fard S, Marrero-Rodriguez F, Kaski JC: Intracoronary versus intravenous
abciximab administration in patients with ST-elevation myocardial infarction
undergoing thrombus aspiration during primary percutaneous coronary
intervention--effects on soluble CD40 ligand concentrations. Atherosclerosis 206:
523-527. 2009
31. Starke A, Wuthrich RP, Waeckerle-Men Y: TGF-beta treatment modulates PD-L1
and CD40 expression in proximal renal tubular epithelial cells and enhances CD8
cytotoxic T-cell responses. Nephron Exp Nephrol 107: e22-29. 2007
32. Li H, Nord EP: IL-8 amplifies CD40/CD154-mediated ICAM-1 production via
the CXCR-1 receptor and p38-MAPK pathway in human renal proximal tubule
cells. Am J Physiol Renal Physiol 296: F438-445. 2009
33. Kairaitis L, Wang Y, Zheng L, Tay YC, Harris DC: Blockade of CD40-CD40
ligand protects against renal injury in chronic proteinuric renal disease. Kidney Int
64: 1265-1272. 2003
34. Nguyen TA, Lordkipanidze M, Diodati JG, Palisaitis DA, Schampaert E, Turgeon
J, Pharand C: Week-long high-maintenance dose clopidogrel regimen achieves
76
better platelet aggregation inhibition than a standard loading dose before
percutaneous coronary intervention: results of a double-blind, randomized clinical
trial. J Interv Cardiol 22: 368-377. 2009
35. Fefer P, Hod H, Hammerman H, Segev A, Beinart R, Boyko V, Behar S,
Matetzky S: Usefulness of pretreatment with high-dose clopidogrel in patients
undergoing primary angioplasty for ST-elevation myocardial infarction. Am J
Cardiol 104: 514-518. 2009
36. Gladding P, Webster M, Zeng I, Farrell H, Stewart J, Ruygrok P, Ormiston J, El-
Jack S, Armstrong G, Kay P, Scott D, Gunes A, Dahl ML: The antiplatelet effect
of higher loading and maintenance dose regimens of clopidogrel: the PRINC
(Plavix Response in Coronary Intervention) trial. JACC Cardiovasc Interv 1: 612-
619. 2008
37. Azar RR, Kassab R, Zoghbi A, Aboujaoude S, El-Osta H, Ghorra P, Germanos
M, Salame E: Effects of clopidogrel on soluble CD40 ligand and on high-
sensitivity C-reactive protein in patients with stable coronary artery disease. Am
Heart J 151: 521.e1-521.e4. 2006
38. Saw J, Madsen EH, Chan S, Maurer-Spurej E: The ELAPSE (Evaluation of Long-
Term Clopidogrel Antiplatelet and Systemic Anti-Inflammatory Effects) study. J
Am Coll Cardiol 52: 1826-1833. 2008
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4.7 Table and Figure Legends
Table 4.1. Baseline Characteristics of the Normal Controls (Normal), Patient Controls
(Control), and the Renal Artery Stenosis Patients (RAS). Values are mean ± SD, or number and percentage of patients. BMI, body mass index; sCD40L, soluble CD40 ligand;
MI, myocardial infarction; NA, not applicable. aNormal vs. RAS. bPatient control vs. RAS
Figure 4-1. Soluble CD40L levels in Normal Controls, Patient Controls, and Renal
Artery Stenosis Patients (RAS). Box plot represents interquartile range with the median value shown as a horizontal bar within each box. Minimum and maximum values are shown in the bars outside each box. *p<0.001 vs Normal Control Subjects.
Figure 4-2. Soluble CD40L levels in patients With Platelet Rich Emboli Captured
Within the filter at Immediate Post Procedure. Analysis of Angioguard contents was performed in 35/39 (90%) of patients randomized to Angioguard. Nine (9) out of 35 patients (26%) had platelet-rich emboli captured. Data presented as mean ± SEM,
*p=0.02 vs Platelet Rich Emboli.
Figure 4-3. Soluble CD40L in patients with atherosclerotic renal artery stenosis randomly assigned to either abciximab, angioguard embolic protection, both, or neither.
Data presented as mean ± SEM, *p<0.01 vs Baseline, #p<0.05 vs Baseline.
78
4.8 Table and Figures
Table 4.1.
79
Figure 4-1.
700 *
* 600
500
400
sCD40L (pg/ml) sCD40L 300
200
100
0 Normal Control Patient Control RAS Patients Subjects (n=30) Subjects (n=30) (n=84)
80
Figure 4-2.
700.0
600.0
500.0
400.0 *
300.0 sCD40L (pg/ml) *
200.0
Platelet Rich Emboli (n=9) No Platelet Rich Emboli (n=26) 100.0
0.0 Baseline Immediate Post Procedure
81
Figure 4-3
82
Chapter 5 - CD40 Mediated Fibrosis in Chronic and Ischemic Renal Disease
5.1 Chronic Kidney Disease and Renal Ischemia
Recent data indicates that chronic kidney disease (CKD) is prevalent, affecting up
to 11% of the US adult population.1 Platelet activation and inflammation have been
implicated in the progression CKD.2 Cardiovascular disease is both common and a major
cause of mortality in patients with CKD.3, 4 This uremic cardiomyopathy is characterized
by a decrease in diastolic function, left ventricular hypertrophy, oxidant stress, and both
cardiac and renal fibrosis.5-7 We have shown that the cardiotonic steroid marinobufagenin
(MBG), signaling through the Na/K-ATPase, causes many of the adverse pathological effects of experimental uremic cardiomyopathy induced by 5/6th nephrectomy (PNx) in the rat. 8 CTS bind to the Na/K-ATPase and convert it into a signal transducer capable of activating multiple protein kinase cascades.9-11 Src binds to the Na/K-ATPase α1 subunit forming a functional signaling complex.12 CTS bind to the Na/K-ATPase and induce a conformational change which activates Src.12 Src transactivates EGFR which results in
the activation of phospholipase C (PLC), phosphoinsitide 3-kinase (PI3K), mitogen- activated protein kinases (MAPKs), protein kinase C (PKC), and the generation of reactive oxygen species (ROS).5, 11 We have demonstrated that pharmacologic
administration of MBG causes cardiac hypertrophy and fibrosis, as seen in patients,
83
whereas active immunization against MBG attenuated this in PNx.7, 8 Additionally, cardiac fibroblasts treated with MBG, at concentrations similar to those reported in experimental and clinical renal failure, has been shown to stimulate collagen production.7
This increase in collagen production appears to be dependent on the Na/K-ATPase-Src-
EGFR-ROS signaling cascade.7 The transcription factor Friend leukemia integration-1
(Fli-1) has been shown to be a negative regulator of collagen synthesis.13, 14 PKC- δ phosphorylates Fli-1 and promotes collagen synthesis.15 We have shown that MBG signaling through the Na/K-ATPase, cause PKC- δ translocation to the nucleus leading to
Fli-1 phosphorylation and collagen production.16
Renal artery stenosis (RAS) is a major cause of renal ischemia affecting 1-5% of the 60 million Americans with hypertension.17-19 Recent data suggests an incidence of up to 7% in patients over the age of 65.20 RAS is a major cause of secondary hypertension and an important cause of renal failure in patients with end stage renal disease. 21-24
Although the clinical utility of stent revascularization in patients with RAS is still uncertain, several studies suggest that at least a portion of patients develop a loss of kidney function post-procedure. 21, 22, 25, 26 Several mechanisms have been implicated as possible causes for a post-procedural decline in renal function such as contrast nephrotoxicity, and atheroembolization. Inflammation, fibrosis, and increases in oxidative stress leading to endothelial dysfunction have also been implicated in decreased renal function in the setting of atherosclerotic RAS.27 Increases in ROS have been reported in the stenotic kidneys from animal models of RAS.28, 29 We have shown that patients with RAS have significantly higher values of plasma MBG compared to healthy
84
control subjects, and patients with coronary atherosclerotic disease indicating that RAS
potentiates MBG release.30
5.2 Platelet Activation, CD40 Signaling, and Fibrosis
Increased platelet activation is associated with a variety of vascular disorders
including acute coronary syndromes, stable coronary artery disease, and restenosis
following percutaneous coronary intervention.31, 32 Platelet activation leads to crucial integrin mediated signaling cascades, which result in stable interactions between platelets and the endothelium as well as activation of glycoprotein IIb/IIIa receptors (primary aggregation receptors), release of alpha and dense granules, and expression and secretion of sCD40L.33, 34 Soluble CD40 ligand has been shown to play a vital role in the immune,
inflammatory, and coagulative responses following injury or stress, and implicated in the
generation of renal fibrosis.35-39 Moreover, high levels of sCD40L correlate with adverse cardiovascular events in patients with unstable coronary syndromes, including atherothrombotic lesions.35, 40-42
CD40, a type-I transmembrane receptor and a member of the tumor necrosis
factor (TNF) receptor superfamily 34, is expressed on a wide range of cells and critically
links thrombosis, inflammation, immunity, and fibrosis. Recent work in renal disease
models suggests that an important mediator of renal fibrosis and inflammatory cell
infiltration is CD40 that resides on the surface of the proximal tubular epithelium.
Specifically, CD40 is upregulated after renal injury 43 and activation of the receptor
85
results in 1) infiltration of inflammatory cells into the interstitium of the kidney through
monocyte chemoattractant protein-1 (MCP-1), and intracellular adhesion molecule-1
(ICAM-1) expression 44, and 2) increases plasminogen activator inhibitor type 1 (PAI-1)
and interstitial fibrosis.45-47 Importantly, Angiotensin II, whose release is increased
during renal ischemia, increases TGF-Β that in turn markedly increases expression of
CD40.47 Finally, CD40 activation increases antigen-specific recognition and killing of tubular epithelial cells by cytotoxic CD8+ T cells.47 Inhibition of CD40 significantly
decreased the severity of renal injury in an animal model of chronic proteinuric renal
disease.48 Our preliminary data shows a substantial increase in sCD40L in patients with
renal artery stenosis compared to normal control subjects, as well as increased expression
of CD40 in kidney tissue derived from PNx animals. We speculate that high levels of
systemic sCD40L travel distally to the kidney and activate CD40 on the proximal tubules
resulting in renal fibrosis.
5.3 Preliminary Data: Clinical trial
5.3.1 Soluble CD40 Ligand Levels in Patients with RAS
One hundred patients enrolled at 7 centers undergoing renal artery stenting were
randomized to an embolic protection device (EPD), or double-blind use of a GPIIb/IIIa inhibitor, Abciximab (the RESIST trial). A detailed description of the clinical trial has been previously reported.49 Plasma levels of sCD40L were measured in all patients with
available baseline blood samples (n=84) using a commercially available kit (R&D
86
Systems). Additional blood samples were collected from 30 healthy volunteers (Normal
Controls), and 30 patients with atherosclerosis, but without renal artery stenosis (Patient
Controls). A detailed description of this study has been reported and is described in
Chapter 4.50 Platelet activation is an important component of the atherosclerotic process
and shedding of sCD40L is a prominent feature of platelet activation. Our data confirms
that normal healthy controls, free of atherosclerosis, have low levels of circulating
sCD40L, whereas older subjects with atherosclerotic RAS are much higher (Figure 5-
1).50 Importantly, the increase in sCD40L is not specific to renal artery stenosis, since a
similar degree of elevation was observed in patients with atherosclerosis without RAS.
700 *
600 *
500
400
sCD40L (pg/ml) sCD40L 300
200
100
0 Normal Control Patient Control RAS Patients Subjects (n=30) Subjects (n=30) (n=84)
Figure 5-1: Soluble CD40 ligand levels in Normal Controls, Patient Controls, and RAS
Patients. *p<0.01 vs. Normal Controls.
87
5.3.2 Circulating concentrations of marinobufagenin (MBG) are substantially increased in patients with ischemic renal disease.
Plasma MBG levels were measured in patients with ischemic renal disease obtained from the aforementioned “RESIST” trial, non-RAS patient controls who were scheduled for coronary angiography, and normal healthy individuals. Marinobufagenin levels were noted to be significantly higher in patients with RAS compared with those of the other 2 groups (Figure 5-2).30 Multivariate analysis shows that occurrence of RAS is independently related to marinobufagenin levels. In addition, renal artery revascularization by stenting partially reversed marinobufagenin levels in the patients with RAS.30
Figure 5-2: MBG levels in Normal Controls, Patient Controls, and RAS Patients.
88
5.3.3 Soluble CD40 concentrations appear to predict outcomes in patients with
ischemic renal disease.
Very limited data is available on the effects of the CD40-sCD40L interactions on clinical outcome, with no published data in patients with ischemic RAS. Lajer et al reported higher levels of sCD40L in type-1 diabetics who developed nephropathy but no increase in mortality or rate of progression to ESRD.51 They had no data on CD40 and outcome. Recently we collaborated with Dr. Phillip Kalra in Manchester UK, assessing
CD40 and sCD40L (measured at the University of Toledo) in a single center cohort of
126 patients with atherosclerotic RAS followed longitudinally. We have observed a statistically significant inverse correlation between baseline circulating levels of CD40
(not tissue bound) and change in GFR over time. Specifically, there was less loss of GFR
(p=0.03) and better survival with higher baseline soluble CD40 (p=0.06). This is in agreement with the hypothesis that soluble CD40 “quenches” sCD40L thereby preventing activation of membrane-bound CD40. We also noted a trend toward increased mortality with higher levels of sCD40L (Figure 5-3).
89
45
40
35
30
25
20
Percent Mortality 15
10
5
0 < 100 sCD40L (pg/ml) 100-300 sCD40L (pg/ml) > 300 sCD40L (pg/ml)
Figure 5-3: Relationship between sCD40L and mortality rates in RAS patients. Note that in patients with high levels of sCD40L, mortality rates reach 40% during the follow up period obtained in the study. Because of the small numbers in this subset, the impressive trend is not statistically significant.
5.4 Preliminary Data: Animal Studies
We subjected Spargue-Dawley rats to either PNx surgery or infusion of MBG through minipumps at a dose designed to achieve similar elevations in plasma MBG as seen with PNx .8 We have previously demonstrated that this infusion of MBG produces substantial renal fibrosis by 4 weeks.6 When we examined the renal cortex of such animals (> 80% proximal tubule by volume), we noted substantial increases in CD40 expression (Figure 5-4, A). Of perhaps greater interest, we found that administration of either spironolactone or antibodies to MBG (either 3E9 mAb or digibind) resulted in marked decreases in renal fibrosis in the PNx model (Figure 5-5 and Figure 5-6, B). Our
90
work describing the use of 3E9 and digibind has recently been reported and is described
in detail in Chapter 2.52 We also noted marked increases in signaling through CD40 as
evidenced by PAI expression (Figure 5-4, B). We next observed that maneuvers which
prevented MBG signaling through the Na/K-ATPase, either administration of antibody
active against MBG or administration of the aldosterone antagonist, spironolactone,
which we have also demonstrated directly inhibits CTS binding to the Na/K-ATPase 53,
resulted in marked decreases in cortical CD40 expression (Figure 5-6, A, and Figure 5-7).
A B Sham PNx MBG Sham PNx MBG CD40 PAI-1
Actin Actin
3.5 2.00 1.80 3 * * 1.60 * ** 2.5 1.40 * 2 1.20
1.00 1.5 0.80 1 0.60
0.5 Expression1 of (Fraction Control) 0.40 - CD40 ExxpressionCD40 of (Fraction Control) PAI 0.20 0 Sham (n=10) PNx (n=10) MBG (n=8) 0.00 Sham (n=6) PNx (n=6) MBG (n=6)
Figure 5-4: Representative Western blot and quantitative data of (A) CD40 and (B) PAI-
1 expression derived form kidney cortex tissue, mean ± s.e.m. Sham (Sham operated
controls); PNx (5/6th nephrectomy); MBG (MBG infusion 10µg/kg/day). *p<0.01 vs.
Sham, **p<0.05 vs. PNx.
91
A B Collagen-1
Sham PNx 3E9 Dig
Sham PNx Actin
2.5 ** ** Dig 3E9 mAb 2 ## 7 * ** 6 # 1.5 5
4 Control) 1
* of (Fraction Expression 1
* - 3 # # 0.5 Fibrosis (% Area) 2 Collagen
1 0 0 Sham (n=6) PNx (n=6) 3E9 (n=6) Dig (n=6) Sham PNx DIG 3E9 mAb
Figure 5-5: Representative serius red fast green staining images (A), and (B) Western blot expression of collagen-1 with quantified data of kidney cortex tissue derived from
Sham, PNx, Digibind (Dig), and 3E9 mAb treated animals, mean ± s.e.m. **p<0.01 vs.
Sham, *p<0.05 vs. Sham, #p<0.01 vs. PNx, ##p<0.05 vs. PNx.
92
A Sham PNx PNx+SP SP B Sham PNx PNx+SP SP CD40 Collagen-1
Actin Actin 3.5 ** ## 3.5 ** 3 ## 3 2.5 ## * 2.5 2 # # 2 * 1.5 1.5 Control)
1 Expression1 of (Fraction - 1
0.5 0.5 CD40 ExpressionCD40 (Fraction of Control) Collagen 0 0 Sham (n=10) PNx (n=10) PNx+SP (n=8) SP (n=8) Sham (n=6) PNx (n=6) PNx + SP SP (n=6) (n=6) Figure 5-6: Representative Western blot and quantified data of (A) CD40, and (B) collagen-1 expression derived from kidney cortex tissue, mean ± s.e.m. Sham (Sham operated controls); PNx (5/6th nephrectomy); PNx+SP (PNx animals treated with spironolactone 80mg/kg/day); SP (Sham animals treated with spironolacton). **p<0.01 vs. Sham, *p<0.05 vs. Sham, #p<0.01 vs. PNx, ##p<0.01 vs. SP
93
Sham PNx 3E9 Dig
CD40
Actin
4 ** 3.5
3 ## * 2.5 * # 2
1.5
1
CD40 ExpressionCD40 (Fraction of Control) 0.5
0 Sham (n=7) PNx (n=6) 3E9 (n=6) Dig (n=6)
Figure 5-7: Representative Western blot and quatitiative data of CD40 expression derived fromkidney cortex tissue, mean ± s.e.m. Sham (Sham operated controls); PNx
(5/6th nephrectomy); 3E9 (PNx animals treated with 3E9 mAb); Dig (PNx animals treated with Digibind). **p<0.01 vs. Sham, *p<0.05 vs. Sham, #p<0.01 vs. PNx, ##p<0.05 vs.
PNx
5.5. Preliminary Data: LLC-PK1 cells
We have shown that MBG treatment in cardiac fibroblasts results in a substantial increase in procollagen-1 expression.7 Using a pig kiney proximal tubual cell line (LLC-
PK1 cells), we have demonstrated that MBG treatment resulted in a significant increase in both CD40 and procollagen-1 expression (Figure 5-8, A and B). Reactive oxygen 94
species (ROS) have been shown to induce CD40 signaling in vascular smooth muscle
cells.54 CTS signaling through the Na/K-ATPase induces ROS production and its
downsteam effects, such as cardiac and renal fibrosis, can be prevented by ROS
scavenging.5 Our collaborators have developed an LLC-PK1 cell line in which the alpha
1 isoform of the Na/K-ATPase (required for CTS signaling) has been knocked down
(PY-17 cells).55 Based on this background, we explored the potential crosstalk between
MBG induced ROS and CD40 expression using the previously described cell lines as
well as treatment with glucose oxidase (GO) which induces sustained levels of H2O2.
MBG and GO treatment resulted in a significant increase in CD40 and procollagen-1
expression, and this effect was midigated by treatment in PY-17 cells (Figures 5-9 and 5-
10). Admittedly, the decrease in procollagen-1 expression in PY-17 cells is more pronounced than the decrease in CD40 expression. Our in vivo results suggest that MBG signaling is required for procollagen-1 production, and contributes to increases in CD40 expression.
95
A B CD40 Procollagen-1
Actin Actin
2.5 3 * * 2.5 2
2 1.5 1.5
1 Control) 1
0.5 0.5 Procollagen Expression Expression of Procollagen (Fraction CD40 Expression (Fraction of Control) 0 0 Control (n=5) MBG 10nM (n=5) Control (n=5) MBG 10nM (n=5)
Figure 5-8: Representative Western blot and quantified data of CD40 expression (A) and procollagen-1 expression (B) from LLC-PK1 cells treated with MBG (10nM) for
24hrs (n=5 experiments). * 96 Control MBG (10nM) GO 1mU GO 3mU LLC PY-17 LLC PY-17 LLC PY-17 LLC PY-17 CD40 Actin 3 * * * # # # 2.5 2 1.5 CD 40 Expression (fraction (fraction control) of 1 0.5 0 LLC-Control PY-17 LLC-MBG PY-17-MBG LLC-GO 1mU PY-17-GO LLC-GO 3mU PY-17-GO Control (10nM) (10nM) 1mU 3mU Figure 5-9: Representative Western blot and quantified data of CD40 expression from LLC-PK1 cells (LLC) and PY-17 cells treated with MBG (10nM) and GO (1 and 3 mU) for 24hrs (n=5 experiments). *p<0.01 vs. Controls, #p<0.05 vs. PY-17-MBG, PY-17-GO 1mU, and PY-17-GO 3mU 97 Control MBG (10nM) GO 1mU GO 3mU LLC PY-17 LLC PY-17 LLC PY-17 LLC PY-17 Procollagen-1 Actin * 3.5 # * * # 3 # 2.5 2 1.5 (fraction of of (fraction control) Procollagen Expression Expression Procollagen 1 0.5 0 LLC-Control PY-17 LLC-MBG PY-17-MBG LLC-GO 1mU PY-17-GO LLC-GO 3mU PY-17-GO Control (10nM) (10nM) 1mU 3mU Figure 5-10: Representative Western blot and quantified data of procollagen-1 expression from LLC-PK1 cells (LLC) and PY-17 cells treated with MBG (10nM) and GO (1 and 3 mU) for 24hrs (n=5 experiments). *p<0.01 vs. Controls, #p<0.01 vs. PY-17- MBG, PY-17-GO 1mU, and PY-17-GO 3mU 98 5.6 Conclusions The CD40/CD40L signaling cascade has been shown to induce inflammation and fibrosis in proximal tubular epithelial cells.39 Specifically, stimulation of the CD40 receptor by sCD40L causes increased expression MCP-1 and the pro-fibrotic mediator PAI-1 leading to the generation of fibrosis.39 Furthermore, inhibition of CD40/CD40L signaling has been shown to decrease the severity of renal injury in an animal model of chronic proteinuric renal disease.48 Our preliminary data from clinical trials demonstrates that 1) plasma levels of sCD40L and MBG are significantly increased in patients with ischemic renal disease, and 2) within these patients levels of circulation CD40 may predict renal function. Our data in animal models suggests that PNx and MBG administration results in significant increases in renal tissue expression of CD40 and PAI- 1 whereas treatment with spironolactone, and antibodies to inhibit CTS signaling (digibind and 3E9) resulted in dramatic decreases in CD40 and collagen expression. Our future work will focus on expanding our preliminary results in patients with ischemic renal disease in order to determine if circulating levels of CD40 and CD40L are predictive of renal function. In addition, we will use genetic manipulation both in vivo and in vitro to knock out the CD40 receptor and knock down the Na/K-ATPase α-1 subunit. These manipulations will allow us to investigate the role of CTS signaling, CD40 signaling, and the generation of renal fibrosis. Our ultimate goal is to provide concrete evidence for our working hypothesis in the pathogenesis of renal fibrosis in the setting of chronic and ischemic renal disease (Figure 5-11). 99 Figure 5-11: Potential scheme for CD40 mediate renal fibrosis in the setting of chronic and ischemic renal disease. In the setting of chronic and ischemic renal injury, increased circulating levels of MBG convert the Na/K-ATPase into a signal transducer generating ROS, which may lead to increased expression of CD40 resulting in pro-inflammatory and pro-fibrotic signaling cascades. CD40 signaling has also been implicated in the generation of ROS. Signaling through the CD40 receptor could be stimulated by high circulating levels of sCD40L. MBG signaling causes translocation of PKC-δ to the nucleus resulting in phosphorylation of FLI-1, and increased collagen expression leading to fibrosis. 100 5.7 References for Chapter 5 1. Kronenberg F. Emerging risk factors and markers of chronic kidney disease progression. Nat Rev Nephrol. 2009;5(12):677-689. 2. Tu X, Chen X, Xie Y, Shi S, Wang J, Chen Y, Li J. Anti-inflammatory renoprotective effect of clopidogrel and irbesartan in chronic renal injury. J Am Soc Nephrol. 2008;19(1):77-83. 3. Chade AR, Lerman A, Lerman LO. Kidney in early atherosclerosis. Hypertension. 2005;45(6):1042-1049. 4. Jaradat MI, Molitoris BA. Cardiovascular disease in patients with chronic kidney disease. Semin Nephrol. 2002;22(6):459-473. 5. Bagrov AY, Shapiro JI, Fedorova OV. Endogenous cardiotonic steroids: physiology, pharmacology, and novel therapeutic targets. Pharmacol Rev. 2009;61(1):9-38. 6. Fedorova LV, Raju V, El-Okdi N, Shidyak A, Kennedy DJ, Vetteth S, Giovannucci DR, Bagrov AY, Fedorova OV, Shapiro JI, Malhotra D. The cardiotonic steroid hormone marinobufagenin induces renal fibrosis: implication of epithelial-to-mesenchymal transition. Am J Physiol Renal Physiol. 2009;296(4):F922-934. 7. Elkareh J, Kennedy DJ, Yashaswi B, Vetteth S, Shidyak A, Kim EG, Smaili S, Periyasamy SM, Hariri IM, Fedorova L, Liu J, Wu L, Kahaleh MB, Xie Z, Malhotra D, Fedorova OV, Kashkin VA, Bagrov AY, Shapiro JI. 101 Marinobufagenin stimulates fibroblast collagen production and causes fibrosis in experimental uremic cardiomyopathy. Hypertension. 2007;49(1):215-224. 8. Kennedy DJ, Vetteth S, Periyasamy SM, Kanj M, Fedorova L, Khouri S, Kahaleh MB, Xie Z, Malhotra D, Kolodkin NI, Lakatta EG, Fedorova OV, Bagrov AY, Shapiro JI. Central role for the cardiotonic steroid marinobufagenin in the pathogenesis of experimental uremic cardiomyopathy. Hypertension. 2006;47(3):488-495. 9. Xie Z, Askari A. Na(+)/K(+)-ATPase as a signal transducer. Eur J Biochem. 2002;269(10):2434-2439. 10. Xie Z, Cai T. Na+-K+--ATPase-mediated signal transduction: from protein interaction to cellular function. Mol Interv. 2003;3(3):157-168. 11. Xie Z. Molecular mechanisms of Na/K-ATPase-mediated signal transduction. Ann N Y Acad Sci. 2003;986:497-503. 12. Tian J, Cai T, Yuan Z, Wang H, Liu L, Haas M, Maksimova E, Huang XY, Xie ZJ. Binding of Src to Na+/K+-ATPase forms a functional signaling complex. Mol Biol Cell. 2006;17(1):317-326. 13. Czuwara-Ladykowska J, Shirasaki F, Jackers P, Watson DK, Trojanowska M. Fli- 1 inhibits collagen type I production in dermal fibroblasts via an Sp1-dependent pathway. J Biol Chem. 2001;276(24):20839-20848. 14. Wang Y, Fan PS, Kahaleh B. Association between enhanced type I collagen expression and epigenetic repression of the FLI1 gene in scleroderma fibroblasts. Arthritis Rheum. 2006;54(7):2271-2279. 102 15. Jinnin M, Ihn H, Yamane K, Mimura Y, Asano Y, Tamaki K. Alpha2(I) collagen gene regulation by protein kinase C signaling in human dermal fibroblasts. Nucleic Acids Res. 2005;33(4):1337-1351. 16. Elkareh J, Periyasamy SM, Shidyak A, Vetteth S, Schroeder J, Raju V, Hariri IM, El-Okdi N, Gupta S, Fedorova L, Liu J, Fedorova OV, Kahaleh MB, Xie Z, Malhotra D, Watson DK, Bagrov AY, Shapiro JI. Marinobufagenin induces increases in procollagen expression in a process involving protein kinase C and Fli-1: implications for uremic cardiomyopathy. Am J Physiol Renal Physiol. 2009;296(5):F1219-1226. 17. Derkx FH, Schalekamp MA. Renal artery stenosis and hypertension. Lancet. 1994;344(8917):237-239. 18. Ram CV. Renovascular hypertension. Cardiol Clin. 1988;6(4):483-508. 19. Vokonas PS, Kannel WB, Cupples LA. Epidemiology and risk of hypertension in the elderly: the Framingham Study. J Hypertens Suppl. 1988;6(1):S3-9. 20. Hansen KJ, Edwards MS, Craven TE, Cherr GS, Jackson SA, Appel RG, Burke GL, Dean RH. Prevalence of renovascular disease in the elderly: a population- based study. J Vasc Surg. 2002;36(3):443-451. 21. Balk E, Raman G, Chung M, Ip S, Tatsioni A, Alonso A, Chew P, Gilbert SJ, Lau J. Effectiveness of management strategies for renal artery stenosis: a systematic review. Ann Intern Med. 2006;145(12):901-912. 22. Olin JW. Survival in atherosclerotic renal artery stenosis: its all about renal function, or is it? Catheter Cardiovasc Interv. 2007;69(7):1048-1049. 103 23. Safian RD, Textor SC. Renal-artery stenosis. N Engl J Med. 2001;344(6):431- 442. 24. Hirsch AT, Haskal ZJ, Hertzer NR, Bakal CW, Creager MA, Halperin JL, Hiratzka LF, Murphy WR, Olin JW, Puschett JB, Rosenfield KA, Sacks D, Stanley JC, Taylor LM, Jr., White CJ, White J, White RA, Antman EM, Smith SC, Jr., Adams CD, Anderson JL, Faxon DP, Fuster V, Gibbons RJ, Hunt SA, Jacobs AK, Nishimura R, Ornato JP, Page RL, Riegel B. ACC/AHA 2005 Practice Guidelines for the management of patients with peripheral arterial disease (lower extremity, renal, mesenteric, and abdominal aortic): a collaborative report from the American Association for Vascular Surgery/Society for Vascular Surgery, Society for Cardiovascular Angiography and Interventions, Society for Vascular Medicine and Biology, Society of Interventional Radiology, and the ACC/AHA Task Force on Practice Guidelines (Writing Committee to Develop Guidelines for the Management of Patients With Peripheral Arterial Disease): endorsed by the American Association of Cardiovascular and Pulmonary Rehabilitation; National Heart, Lung, and Blood Institute; Society for Vascular Nursing; TransAtlantic Inter-Society Consensus; and Vascular Disease Foundation. Circulation. 2006;113(11):e463-654. 25. Levin A, Linas S, Luft FC, Chapman AB, Textor S. Controversies in renal artery stenosis: a review by the American Society of Nephrology Advisory Group on Hypertension. Am J Nephrol. 2007;27(2):212-220. 26. Cooper CJ, Murphy TP, Matsumoto A, Steffes M, Cohen DJ, Jaff M, Kuntz R, Jamerson K, Reid D, Rosenfield K, Rundback J, D'Agostino R, Henrich W, 104 Dworkin L. Stent revascularization for the prevention of cardiovascular and renal events among patients with renal artery stenosis and systolic hypertension: rationale and design of the CORAL trial. Am Heart J. 2006;152(1):59-66. 27. Lerman LO, Textor SC, Grande JP. Mechanisms of tissue injury in renal artery stenosis: ischemia and beyond. Prog Cardiovasc Dis. 2009;52(3):196-203. 28. Chade AR, Rodriguez-Porcel M, Herrmann J, Zhu X, Grande JP, Napoli C, Lerman A, Lerman LO. Antioxidant intervention blunts renal injury in experimental renovascular disease. J Am Soc Nephrol. 2004;15(4):958-966. 29. Zhu XY, Chade AR, Rodriguez-Porcel M, Bentley MD, Ritman EL, Lerman A, Lerman LO. Cortical microvascular remodeling in the stenotic kidney: role of increased oxidative stress. Arterioscler Thromb Vasc Biol. 2004;24(10):1854- 1859. 30. Tian J, Haller S, Periyasamy S, Brewster P, Zhang H, Adlakha S, Fedorova OV, Xie ZJ, Bagrov AY, Shapiro JI, Cooper CJ. Renal ischemia regulates marinobufagenin release in humans. Hypertension.56(5):914-919. 31. Santilli F, Basili S, Ferroni P, Davi G. CD40/CD40L system and vascular disease. Intern Emerg Med. 2007;2(4):256-268. 32. Turker S, Guneri S, Akdeniz B, Ozcan MA, Baris N, Badak O, Kirimli O, Yuksel F. Usefulness of preprocedural soluble CD40 ligand for predicting restenosis after percutaneous coronary intervention in patients with stable coronary artery disease. Am J Cardiol. 2006;97(2):198-202. 33. Davi G, Patrono C. Platelet activation and atherothrombosis. N Engl J Med. 2007;357(24):2482-2494. 105 34. Antoniades C, Bakogiannis C, Tousoulis D, Antonopoulos AS, Stefanadis C. The CD40/CD40 ligand system: linking inflammation with atherothrombosis. J Am Coll Cardiol. 2009;54(8):669-677. 35. Mason PJ, Chakrabarti S, Albers AA, Rex S, Vitseva O, Varghese S, Freedman JE. Plasma, serum, and platelet expression of CD40 ligand in adults with cardiovascular disease. Am J Cardiol. 2005;96(10):1365-1369. 36. Inwald DP, McDowall A, Peters MJ, Callard RE, Klein NJ. CD40 is constitutively expressed on platelets and provides a novel mechanism for platelet activation. Circ Res. 2003;92(9):1041-1048. 37. Chakrabarti S, Varghese S, Vitseva O, Tanriverdi K, Freedman JE. CD40 ligand influences platelet release of reactive oxygen intermediates. Arterioscler Thromb Vasc Biol. 2005;25(11):2428-2434. 38. Henn V, Slupsky JR, Grafe M, Anagnostopoulos I, Forster R, Muller-Berghaus G, Kroczek RA. CD40 ligand on activated platelets triggers an inflammatory reaction of endothelial cells. Nature. 1998;391(6667):591-594. 39. Pontrelli P, Ursi M, Ranieri E, Capobianco C, Schena FP, Gesualdo L, Grandaliano G. CD40L proinflammatory and profibrotic effects on proximal tubular epithelial cells: role of NF-kappaB and lyn. J Am Soc Nephrol. 2006;17(3):627-636. 40. Nannizzi-Alaimo L, Rubenstein MH, Alves VL, Leong GY, Phillips DR, Gold HK. Cardiopulmonary bypass induces release of soluble CD40 ligand. Circulation. 2002;105(24):2849-2854. 106 41. Kritharides L, Lau GT, Freedman B. Soluble CD40 ligand in acute coronary syndromes. N Engl J Med. 2003;348(25):2575-2577; author reply 2575-2577. 42. Burdon KP, Langefeld CD, Beck SR, Wagenknecht LE, Carr JJ, Rich SS, Freedman BI, Herrington D, Bowden DW. Variants of the CD40 gene but not of the CD40L gene are associated with coronary artery calcification in the Diabetes Heart Study (DHS). Am Heart J. 2006;151(3):706-711. 43. Gaweco AS, Mitchell BL, Lucas BA, McClatchey KD, Van Thiel DH. CD40 expression on graft infiltrates and parenchymal CD154 (CD40L) induction in human chronic renal allograft rejection. Kidney Int. 1999;55(4):1543-1552. 44. Li H, Nord EP. IL-8 amplifies CD40/CD154-mediated ICAM-1 production via the CXCR-1 receptor and p38-MAPK pathway in human renal proximal tubule cells. Am J Physiol Renal Physiol. 2009;296(2):F438-445. 45. Pontrelli P, Rossini M, Infante B, Stallone G, Schena A, Loverre A, Ursi M, Verrienti R, Maiorano A, Zaza G, Ranieri E, Gesualdo L, Ditonno P, Bettocchi C, Schena FP, Grandaliano G. Rapamycin inhibits PAI-1 expression and reduces interstitial fibrosis and glomerulosclerosis in chronic allograft nephropathy. Transplantation. 2008;85(1):125-134. 46. Rerolle JP, Hertig A, Nguyen G, Sraer JD, Rondeau EP. Plasminogen activator inhibitor type 1 is a potential target in renal fibrogenesis. Kidney Int. 2000;58(5):1841-1850. 47. Starke A, Wuthrich RP, Waeckerle-Men Y. TGF-beta treatment modulates PD-L1 and CD40 expression in proximal renal tubular epithelial cells and enhances CD8 cytotoxic T-cell responses. Nephron Exp Nephrol. 2007;107(1):e22-29. 107 48. Kairaitis L, Wang Y, Zheng L, Tay YC, Harris DC. Blockade of CD40-CD40 ligand protects against renal injury in chronic proteinuric renal disease. Kidney Int. 2003;64(4):1265-1272. 49. Cooper CJ, Haller ST, Colyer W, Steffes M, Burket MW, Thomas WJ, Safian R, Reddy B, Brewster P, Ankenbrandt MA, Virmani R, Dippel E, Rocha-Singh K, Murphy TP, Kennedy DJ, Shapiro JI, D'Agostino RD, Pencina MJ, Khuder S. Embolic protection and platelet inhibition during renal artery stenting. Circulation. 2008;117(21):2752-2760. 50. Haller S, Adlakha S, Reed G, Brewster P, Kennedy D, Burket MW, Colyer W, Yu H, Zhang D, Shapiro JI, Cooper CJ. Platelet activation in patients with atherosclerotic renal artery stenosis undergoing stent revascularization. Clin J Am Soc Nephrol.6(9):2185-2191. 51. Lajer M, Tarnow I, Michelson AD, Jorsal A, Frelinger AL, Parving HH, Rossing P, Tarnow L. Soluble CD40 ligand is elevated in type 1 diabetic nephropathy but not predictive of mortality, cardiovascular events or kidney function. Platelets. 2010;21(7):525-532. 52. Haller ST, Kennedy DJ, Shidyak A, Budny GV, Malhotra D, Fedorova OV, Shapiro JI, Bagrov AY. Monoclonal Antibody Against Marinobufagenin Reverses Cardiac Fibrosis in Rats With Chronic Renal Failure. Am J Hypertens. March 2012 [epub ahead of print]. 53. Tian J, Shidyak A, Periyasamy SM, Haller S, Taleb M, El-Okdi N, Elkareh J, Gupta S, Gohara S, Fedorova OV, Cooper CJ, Xie Z, Malhotra D, Bagrov AY, 108 Shapiro JI. Spironolactone attenuates experimental uremic cardiomyopathy by antagonizing marinobufagenin. Hypertension. 2009;54(6):1313-1320. 54. Souza HP, Frediani D, Cobra AL, Moretti AI, Jurado MC, Fernandes TR, Cardounel AJ, Zweier JL, Tostes RC. Angiotensin II modulates CD40 expression in vascular smooth muscle cells. Clinical Science. 2009;116:423-431. 55. Liang M, Cai T, Tian J, Qu W, Xie Z. Functional characturization of Src- interacting Na/K-ATPase using RNA interference assay. J Biol Chem. 2006;281:19709-19719. 109 Chapter 6 – Summary and Conclusions 6.1 – Immunization As A Potential Therapy For Uremic Cardiomyopathy Uremic cardiomyopathy is characterized as chronic renal failure accompanied with the development of severe cardiovascular disease, which ultimately accounts for the high morbidity and mortality in patients with this disease state.1 We have shown that the cardiotonic steroid, MBG signaling through the Na/K-ATPase significantly contributes to the development of experimental uremic cardiomyopathy induced by 5/6th nephrectomy (PNx) in the rat.2, 3 Specifically, we have demonstrated that PNx animals develop diastolic dysfunction, cardiac hypertrophy, cardiac and renal fibrosis, elevated levels of cardiac and systemic oxidative stress, and elevated circulating levels of MBG.2, 3 Chronic administration of MBG in normotensive rats (at a concentration similar to values reported in PNx animals) results in a similar cardiac phenotype as seen in PNx.2 We have demonstrated that active immunization against MBG in PNx animals attenuates these effects.2 In Chapter 2 we report that passive immunization with a single dose of an anti- MBG antibody (3E9 mAb) during the fifth week following PNx surgery drastically reduced systolic BP, cardiac fibrosis, and cardiac levels of oxidative stress. We also demonstrated that 3E9 treatment increased cardiac levels of Fli-1, a negative regulator of collagen synthesis. Our results indicate that immunization against MBG may provide a potential therapy for uremic cardiomyopathy. 110 6.2 – Treatment With Rapamycin As A Potential Therapy For Uremic Cardiomyopathy The mTOR pathway has been implicated in the progression of many different forms of renal disease including experimentally induced uremic cardiomyopathy.4, 5 Treatment with rapamycin (an mTOR inhibitor) has been shown to attenuate inflammation, fibrosis, and cardiac hypertrophy in experimental models of renal disease.4 In Chapter 3 we demonstrate that treatment with rapamycin in PNx animals significantly reduced cardiac fibrosis. Additionally, we were able to show that treatment with rapamycin in cardiac fibroblasts drastically reduced MBG induced collagen production when co-administered with MBG at concentrations similar to those reported in experimental and clinical renal failure. The biosynthesis of MBG is currently under debate. In toads, MBG has been postulated to be synthesized from cholesterol via a bile acid pathway form cholanic acids.1 In addition to acting as an mTOR inhibitor, rapamycin also acts as a competitive inhibitor of CYP27A1, a key rate-limiting enzyme of the bile acid pathway.6 Rapamycin treatment significantly reduced circulating levels of MBG in PNx animals. Treatment with rapamycin was also shown to drastically reduce MBG levels in human chorionic epithelial cells (JEG-3 cells), which produce MBG, by 52%. Our data demonstrates that rapamycin may offer a potential therapy for uremic cardiomyopathy acting as both an anti-fibrotic agent and a potential inhibitor of MBG production. 111 6.3 – Platelet Activation and CD40 Signaling in Chronic and Ischemic Renal Disease Increased platelet activation is associated with a variety of vascular disorders including acute coronary syndromes, stable coronary artery disease, and restenosis following percutaneous coronary intervention. 7, 8 Soluble CD40L is a particularly attractive marker for platelet activation since it is shed from the surface of activated platelets, is easily measured, and meaningfully participates in a number of important biologic processes including activation of immunity and thrombosis.9 In Chapter 4 we report that patients with renal artery stenosis have significantly high levels of circulating sCD40L compared to normal control subjects. However, this appears to be a non- specific association with atherosclerosis in general as opposed to being attributable to RAS specifically. More importantly though increased levels of sCD40L prior to the procedure were more likely to have embolization of platelet-rich thrombi and these patients had persistently elevated levels of sCD40L after the procedure. This finding may represent a potentially modifiable feature denoting increased risk for patients referred for renal artery revascularization. CD40, a type-I transmembrane receptor and a member of the tumor necrosis factor (TNF) receptor superfamily, is expressed on a wide range of cells and critically links thrombosis, inflammation, immunity, and fibrosis.9 Recent work in renal disease models suggests that an important mediator of renal fibrosis and inflammatory cell infiltration is CD40 that resides on the surface of the proximal tubular epithelium. Specifically, stimulation of the CD40 receptor by sCD40L causes increased expression MCP-1 and the pro-fibrotic mediator PAI-1 leading to the generation of fibrosis.10 Our 112 preliminary data in animal models suggests that partial nephrectomy and MBG administration results in significantly increased expression of CD40 in the renal cortex tissue derived from these animals (Chapter 5). We also show increased expression of PAI-1 indicating an increase in CD40 signaling within the kidney (Chapter 5). Additionally, we provide evidence that maneuvers aimed at inhibiting cardiotonic steroid signaling through the Na/K-ATPase (spironolactone, 3E9, and digibind treatment) also resulted in a significant decrease in CD40 expression and renal fibrosis (Chapter 5). We speculate that in the setting of ischemic and chronic renal disease, increased circulating levels of sCD40L travel distally to the kidney, and activate the CD40 receptor resulting in the generation of renal fibrosis in a process potentiated by cardiotonic steroid signaling through the Na/K-ATPase. Future work will focus on determining a direct link between cardiotonic steroid signaling, CD40 signaling, and the development of renal fibrosis. 113 6.4 References for Summary and Conclusions 1. Bagrov AY, Shapiro JI, Fedorova OV. Endogenous cardiotonic steroids: physiology, pharmacology, and novel therapeutic targets. Pharmacol Rev. 2009;61(1):9-38. 2. Kennedy DJ, Vetteth S, Periyasamy SM, Kanj M, Fedorova L, Khouri S, Kahaleh MB, Xie Z, Malhotra D, Kolodkin NI, Lakatta EG, Fedorova OV, Bagrov AY, Shapiro JI. Central role for the cardiotonic steroid marinobufagenin in the pathogenesis of experimental uremic cardiomyopathy. Hypertension. 2006;47(3):488-495. 3. Elkareh J, Kennedy DJ, Yashaswi B, Vetteth S, Shidyak A, Kim EG, Smaili S, Periyasamy SM, Hariri IM, Fedorova L, Liu J, Wu L, Kahaleh MB, Xie Z, Malhotra D, Fedorova OV, Kashkin VA, Bagrov AY, Shapiro JI. Marinobufagenin stimulates fibroblast collagen production and causes fibrosis in experimental uremic cardiomyopathy. Hypertension. 2007;49(1):215-224. 4. Lieberthal W, Levine JS. The role of the mammalian target of rapamycin (mTOR) in renal disease. J Am Soc Nephrol. 2009;20(12):2493-2502. 5. Siedlecki AM, Jin X, Muslin AJ. Uremic cardiac hypertrophy is reversed by rapamycin but not by lowering of blood pressure. Kidney Int. 2009;75(8):800- 808. 6. Gueguen Y, Ferrari L, Souidi M, Batt AM, Lutton C, Siest G, Visvikis S. Compared effect of immunosuppressive drugs cyclosporine A and rapamycin on 114 cholesterol homeostasis key enzymes CYP27A1 and HMG-CoA reductase. Basic Clin Pharmacol Toxicol. 2007;100(6):392-397. 7. Santilli F, Basili S, Ferroni P, Davi G. CD40/CD40L system and vascular disease. Intern Emerg Med. 2007;2(4):256-268. 8. Turker S, Guneri S, Akdeniz B, Ozcan MA, Baris N, Badak O, Kirimli O, Yuksel F. Usefulness of preprocedural soluble CD40 ligand for predicting restenosis after percutaneous coronary intervention in patients with stable coronary artery disease. Am J Cardiol. 2006;97(2):198-202. 9. Antoniades C, Bakogiannis C, Tousoulis D, Antonopoulos AS, Stefanadis C. The CD40/CD40 ligand system: linking inflammation with atherothrombosis. J Am Coll Cardiol. 2009;54(8):669-677. 10. Pontrelli P, Ursi M, Ranieri E, Capobianco C, Schena FP, Gesualdo L, Grandaliano G. CD40L proinflammatory and profibrotic effects on proximal tubular epithelial cells: role of NF-kappaB and lyn. J Am Soc Nephrol. 2006;17(3):627-636. 115